Colorimetric and fluorescent proteins

ABSTRACT

The invention relates to intracellular lipid binding proteins that bind retinoids and/or dye ligands and that are modified to transmit or emit light at a variety of different wavelengths.

This application claims benefit of the priority filing date of U.S.Provisional Application Ser. No. 61/340,831, filed Mar. 23, 2010, thecontents of which are specifically incorporated herein by reference intheir entirety.

GOVERNMENT SUPPORT

This invention described herein was made support from the United StatesGovernment under grant number NIH R01 GM067311, awarded by the NationalInstitutes of Health. The United States government has rights in theinvention.

BACKGROUND OF THE INVENTION

The complex functions of biological molecules are difficult toascertain, in part because it is difficult to observe these moleculeswithin functioning biological systems. Thus, the locus of abiomolecule's activity and the factors that actually interact with thebiomolecule may not be apparent because it is difficult to distinguishone biomolecule from another within a living cell or tissue.

Labeled antibodies have been employed to identify specific factorswithin cells and tissues. But antibodies are large molecules that do notreadily penetrate cells and binding of antibodies can often inhibit ormodulate the functioning of the molecule to which it is bound.

Dyes have also been used to ‘color’ different cells and cellularfactors. But researchers may not be able to distinguish one biomoleculefrom another, or trace the activity and functioning of a particularbiomolecule, when using dyes because those dyes generally color manycellular structures and/or interrupt the functioning of the cells and/orbiomolecules of interest.

Labeled antibodies and dyes also fail to provide sufficient signalstrength to permit real-time observation of biomolecule activity. Forexample, while green fluorescent protein (GFP) has been used to observethe location of particular biomolecules within cells and/or tissues, GFPcan require several hours to manifest fluorescence. Hence, the movementsand interactions of GFP-linked biomolecules cannot be adequately tracedin dynamic in vivo systems. Moreover, GFP cannot be used to observeseveral factors or biomolecules at once because GFP emits only onefluorescence color (green) and cannot be used to distinguish onebiomolecule from another. GFP also requires oxygen, which is either notavailable or not plentiful in many cell types.

Therefore, new tools are needed that will permit real-time visualizationof multiple biomolecules and factors at once.

SUMMARY OF THE INVENTION

The invention relates to modified proteins in the intracellular lipidbinding protein (iLBP) family that are characterized by largehydrophobic internal binding cavities and that specifically bind avariety of ligands as protonated Schiff bases with high affinity. TheseiLBP proteins have been modified such that the absorbance and lighttransmission of a chromophore ligand (e.g., a retinoid or dye) can bemodulated across the visual range and into the near infrared range.These proteins are remarkably stable and can be recombinantly generatedand expressed as fusion proteins.

One aspect of the invention is an isolated nucleic acid encoding amodified polypeptide that is a member of the intracellular lipid bindingprotein (iLBP) family, wherein the modified polypeptide transmits oremits light when bound to a retinoid or fluorescent dye molecule, andwherein the intracellular lipid binding protein has been modified sothat an amino acid at any of positions 102-135 can form a Schiff basewith a retinoid (e.g., retinal). In some embodiments, the retinoid or afluorescent dye binds specifically to the modified iLBP protein andforms a Schiff base upon binding. For example, such an isolated nucleicacid can encode a modified polypeptide that has been modified byreplacement of the amino acid at any of positions 102-135 with a lysine.Such a Schiff base-forming iLBP polypeptide can be further modified toinclude amino acid substitutions at a variety of positions to modulatethe light transmission/emission properties of the modifiedpolypeptide:retinoid/dye complex. In fact, as illustrated herein, by avariety of amino acid substitutions can be made to yield iLBPpolypeptides that transmit or emit light over the entire visiblespectrum of light.

In some embodiments, the isolated nucleic acid encodes a modifiedpolypeptide that has been modified by replacement of a glutamine at anyof amino acid positions 107, 108 or 109 with a lysine. In otherembodiments, the isolated nucleic acid encodes a modified polypeptidethat has been modified by replacement of an arginine at any of aminoacid positions 110, 111 or 112 with a lysine. In further embodiments,the isolated nucleic acid can encode a modified polypeptide that hasbeen modified by replacement of an arginine at any of amino acidpositions 131, 132 or 133 with a lysine. In another embodiment, theisolated nucleic can encode a modified intracellular lipid bindingprotein that is modified by replacement of a lysine at any of amino acidpositions 39, 40 or 41 with a leucine, serine or asparagine. In otherembodiments, the isolated nucleic acid can encode a modifiedintracellular lipid binding protein that is modified by replacement ofan arginine at any of amino acid positions 131, 132 or 133 with aglutamine. In further embodiments, the isolated nucleic acid can encodea modified intracellular lipid binding protein that is modified byreplacement of a threonine at any of amino acid positions 50, 51, 52,53, 54 or 55 with an aspartic acid, asparagine, cysteine or a valine. Inanother embodiment, the isolated nucleic acid can encode a modifiedintracellular lipid binding protein that is modified by replacement of atyrosine at any of amino acid positions 59, 60 or 61 with a tryptophan,histidine, threonine, asparagine or phenylalanine. In other embodiments,the isolated nucleic acid can encode a modified intracellular lipidbinding protein that is modified by replacement of an arginine at any ofamino acid positions 57, 58, 59 or 60 with a phenylalanine, tyrosine,tryptophan, leucine, glutamine, glutamic acid, aspartic acid or alanine.In a further embodiment, the isolated nucleic acid can encode a modifiedintracellular lipid binding protein that is modified by replacement of atyrosine at any of amino acid positions 133, 134 or 135 with aphenylalanine. In another embodiment, the isolated nucleic acid canencode a modified intracellular lipid binding protein that is modifiedby replacement of a threonine at any of amino acid positions 28, 29 or30 with a leucine, tryptophan, glutamic acid or aspartic acid. In afurther embodiment, the isolated nucleic acid can encode a modifiedintracellular lipid binding protein that is modified by replacement ofan alanine at any of amino acid positions 30, 31, 32 or 33 with atryptophan, phenylalanine, tyrosine, serine, histidine, glutamic acid orleucine. In other embodiments, the isolated nucleic acid can encode amodified intracellular lipid binding protein that is modified byreplacement of a tyrosine at any of amino acid positions 18, 19 or 20with a tryptophan or phenyalanine. In further embodiments, the isolatednucleic acid can encode a modified intracellular lipid binding proteinthat is modified by replacement of a glutamine at any of amino acidpositions 3, 4 or 5 with an arginine, asparagine, phenylalanine,leucine, alanine, tryptophan, threonine, glutamic acid, histidine, orlysine. In another embodiment, the isolated nucleic acid can encode amodified intracellular lipid binding protein that is modified byreplacement of a methionine at any of amino acid positions 92, 93 or 94with a leucine. In other embodiments, the isolated nucleic acid canencode a modified intracellular lipid binding protein that is modifiedby replacement of a glutamic acid at any of amino acid positions 72, 73or 74 with an alanine or leucine. In other embodiments, the isolatednucleic acid can encode a modified intracellular lipid binding proteinthat is modified by replacement of a glutamine at any of amino acidpositions 36, 37 or 38 with a leucine, methionine or tryptophan. Inother embodiments, the isolated nucleic acid can encode a modifiedintracellular lipid binding protein that is modified by replacement of aglutamine at any of amino acid positions 128, 129 or 130 with n leucine,lysine, glutamic acid or tryptophan.

In some embodiments, the isolated nucleic acid encodes a modifiedintracellular lipid binding protein that is a modified cellular retinoicacid binding protein II (CRABPII) or a modified cellular retinol bindingprotein II (CRBPII).

Another aspect of the invention is a modified intracellular lipidbinding protein (iLBP) that transmits or emits light when bound to aretinoid or fluorescent dye molecule, wherein the intracellular lipidbinding protein has been modified so that an amino acid at any ofpositions 102-135 can form a Schiff base with a retinoid. Such amodified iLBP polypeptide can be modified by replacement of the aminoacid at any of positions 102-135 with a lysine. In some embodiments, themodified intracellular lipid binding protein is a modified cellularretinoic acid binding protein II (CRABPII) or a modified cellularretinol binding protein II (CRBPII).

The modified iLBP polypeptide can be modified by replacement of aglutamine at any of amino acid positions 107, 108 or 109 with a lysine.In other embodiments, the modified iLBP polypeptide can be modified byreplacement of an arginine at any of amino acid positions 110, 111 or112 with a lysine. In further embodiments, the modified iLBP polypeptidecan be modified by replacement of an arginine at any of amino acidpositions 131, 132 or 133 with a lysine. In another embodiment, themodified iLBP polypeptide can be modified by replacement of a lysine atany of amino acid positions 39, 40 or 41 with a leucine, serine orasparagine. In other embodiments, the modified iLBP polypeptide can bemodified by replacement of an arginine at any of amino acid positions131, 132 or 133 with a glutamine. In further embodiments, the modifiediLBP polypeptide can be modified by replacement of a threonine at any ofamino acid positions 50, 51, 52, 53, 54 or 55 with an aspartic acid,asparagine, cysteine or a valine. In another embodiment, the modifiediLBP polypeptide can be modified by replacement of a tyrosine at any ofamino acid positions 59, 60 or 61 with a tryptophan, histidine,threonine, asparagine or phenylalanine. In other embodiments, themodified iLBP polypeptide can be modified by replacement of an arginineat any of amino acid positions 57, 58, 59 or 60 with a phenylalanine,tyrosine, tryptophan, leucine, glutamine, glutamic acid, aspartic acidor alanine. In a further embodiment, the modified iLBP polypeptide canbe modified by replacement of a tyrosine at any of amino acid positions133, 134 or 135 with a phenylalanine. In another embodiment, themodified iLBP polypeptide can be modified by replacement of a threonineat any of amino acid positions 28, 29 or 30 with a leucine, tryptophan,glutamic acid or aspartic acid. In a further embodiment, the modifiediLBP polypeptide can be modified by replacement of an alanine at any ofamino acid positions 30, 31, 32 or 33 with a tryptophan, phenylalanine,tyrosine, serine, histidine, glutamic acid or leucine. In otherembodiments, the modified iLBP polypeptide can be modified byreplacement of a tyrosine at any of amino acid positions 18, 19 or 20with a tryptophan or phenyalanine. In further embodiments, the modifiediLBP polypeptide can be modified by replacement of a glutamine at any ofamino acid positions 3, 4 or 5 with an arginine, asparagine,phenylalanine, leucine, alanine, tryptophan, threonine, glutamic acid,histidine, or lysine. In another embodiment, the modified iLBPpolypeptide can be modified by replacement of a methionine at any ofamino acid positions 92, 93 or 94 with a leucine. In other embodiments,the modified iLBP polypeptide can be modified by replacement of aglutamic acid at any of amino acid positions 72, 73 or 74 with analanine or leucine. In other embodiments, the modified iLBP polypeptidecan be modified by replacement of a glutamine at any of amino acidpositions 36, 37 or 38 with a leucine, methionine or tryptophan. Inother embodiments, the modified iLBP polypeptide can be modified byreplacement of a glutamine at any of amino acid positions 128, 129 or130 with a leucine, lysine, glutamic acid or tryptophan.

In some embodiments, the modified polypeptide comprises an amino acidsequence selected from the group consisting of SEQ ID NO:6-28, 39-47, ora combination thereof.

Another aspect of the invention is a hybrid nucleic acid comprising anisolated (modified) iLBP nucleic acid joined to a fusion partner nucleicacid that encodes a fusion partner polypeptide. In such a hybrid nucleicacid, the isolated (modified) iLBP nucleic acid can be joined in frameto the fusion partner nucleic acid.

Another aspect of the invention is a fusion protein comprising amodified iLBP of the invention joined to a fusion partner polypeptide.In such a fusion protein, the modified iLBP of the invention can bejoined in frame to the fusion partner.

Another aspect of the invention is an expression cassette comprising anisolated (modified) iLBP nucleic acid of the invention and at least onenucleic acid segment encoding a regulatory element.

Another aspect of the invention is a vector comprising an isolated(modified) iLBP nucleic acid of the invention. In some embodiments, thevector comprised an expression cassette comprising an isolated(modified) iLBP nucleic acid of the invention and at least one nucleicacid segment encoding a regulatory element.

Another aspect of the invention is a host cell comprising an isolated(modified) iLBP nucleic acid of the invention and at least one nucleicacid segment encoding a regulatory element. In some embodiments, theisolated nucleic acid within the host cell is within an expressioncassette, a vector or a combination thereof.

Another aspect of the invention is a method of observing a targetprotein in vivo comprising contacting a living cell with a retinoid ordye that binds a modified polypeptide encoded by the isolated nucleicacid of claim 1, wherein the cell expresses a fusion protein comprisingthe modified polypeptide fused in frame with the target protein.

DESCRIPTION OF THE FIGURES

FIG. 1A illustrates the range of light colors that the modified CRBPIIproteins described herein transmit. Nucleic acids encoding modifiedCRBPII polypeptides were expressed in E. coli and purified by ionexchange chromatography. Retinal was added to the purified proteins, andabsorption spectra were taken of each purified protein in solution. Thehuman CRBPII polypeptides shown have the following modifications andmaximum wavelengths of absorption: Q108K:T51D (λmax=474 nm);Q108K:K40L:Y60W (λmax=512 nm); Q108K:K40L:R58F (λmax=524 nm);Q108K:K40L:R58Y (λmax=535 nm); Q108K:K40L:R58Y,T51V (λmax=563);Q108K:K40L:R58W:T51V:T53C (λmax=585 nm);108K:K40L:R58W:T51V:T53C:T291L:Y19W (λmax=591 nm);Q108K:K40L:R58W:T51V:T53C:T29L:Y19W:Q4W (λmax=613 nm);Q108K:K40L:R58W:T51V:T53C:T29L:Y19W:Q4R:A33W (λmax=644 nm). A shorthandnotation is used throughout the application for describing modificationswhere the first letter identifies the amino acid that is naturallypresent in the polypeptide, number is the position of that amino acid inthe polypeptide and the following letter identifies the amino acid thatreplaced the natural amino acid. Amino acids are identified by theirsingle letter amino acid designations. FIG. 1B shows modified CRBPIIpolypeptides bound to retinal when loaded onto an anion-exchange column,illustrating that the modified CRBPII polypeptides can be used ascolorimetric tags for protein purification.

FIG. 1B shows various CRBPII modified polypeptides bound to retinal thatwere loaded onto an anion-exchange column. This figure illustrates thatthe modified CRBPII polypeptides described herein can be used as acolorimetric tag for protein purification.

FIG. 2 illustrates colorimetric detection of modified CRBPIIpolypeptides within bacterial cells. Modified CRBPII polypeptides wereexpressed in E. coli, retinal was added to the cells, and the cells werespun down to show the variously colored cell pellets resulting fromexpression of the various colored proteins.

FIG. 3A-C illustrates in vivo visualization of CRBP fluorescence in E.coli cells using a fluorescence microscope (400× magnification) with ared filter. FIG. 3A shows wild type cells treated with merocyanine dyeligand (no CRBP protein is present in these cells). FIG. 3B shows E.coli cells expressing wild-type human CRBPII, which does not bind thefluorescent ligand. Although the merocyanine dye was added to the cells,it does not form a complex with the wild-type CRBP and no fluorescenceis observed. FIG. 3C shows E. coli cells expressing modified humanCRBPII treated with merocyanine dye ligand. The modified human CRBPIIpolypeptide binds the merocyanine dye and fluorescence within the cellsis clearly visible.

FIG. 4A-C illustrates the in vivo fluorescence of modified CRBP fusionproteins in the presence of a merocyanine dye ligand. FIG. 4A is aschematic diagram of the fusion proteins used to conduct the experimentsin FIGS. 4B and 4C, respectively. FIG. 4B shows confocal micrographs ofhuman osteosarcoma cells transfected with pEGFP-CRBP vector, where themerocyanine dye was added. As shown, the GFP-CRBP fusion product isexpressed and fluorescence is detected throughout the cell from both theGFP (left panel: Excitation with blue light) and the CRBP segment(middle panel: excitation with 594 nm light). The right panel shows andoverlay of green and red and bright-field pictures, further illustratingthat the GFP and CRBP fluorescence co-localizes. FIG. 4C shows confocalmicrograph of human osteosarcoma cells transfected with pEGFP-CRBP-RBvector, where the merocyanine dye was also present. RB (retinoblastomaprotein) directs the protein complex of GFP-CRBP-RB to the nucleus.Thus, the GFP-CRBP-RB fusion product is expressed and localized in thenucleus as shown in FIG. 4C. Fluorescence is detected in the nucleus ofthe cell from both the GFP (left panel: Excitation with blue light) andthe CRBP polypeptide segments (middle panel: excitation with 594 nm).The right panel shows and overlay of green and red and bright-fieldpictures, further illustrating that the GFP and CRBP fluorescenceco-localizes. Note also that while the merocyanine dye is likely presentthroughout the cell, the fluorescent signal is observed only within thecell nuclei, indicating that binding between the CRBP polypeptide andthe dye is needed to generate a signal.

FIG. 5A-C show that modified CRABPII polypeptides can work asfluorescence-based pH sensors when combined with a fluorescentmerocyanine dye. FIG. 5A shows absorption spectra taken over a widerange of pH conditions. FIG. 5B shows a titration curve made from thedata provided in FIG. 5A, illustrating that light absorption varies withpH. As shown, the smallest absorption corresponds to the highest pH andthe lowest absorption corresponds to the lowest pH. FIG. 5C showsfluorescence spectra of a mutant CRABPII/merocyanine dye complex at pH7.3 (the highest emission) and at pH 8.6 (the lowest emission), thestructure of the associated pH sensitive merocyanine dye upon formationof the Schiff base with the protein is shown below.

FIG. 6A-D illustrates the light absorption and transmission propertiesof two modified CRABPII polypeptides in the presence of retinal at pH5.0 and pH 7.3. FIG. 6A shows that the first modified CRABPIIpolypeptide (SEQ ID NO:43) has a darker color (blue when seen in color)at pH 5.0 and a lighter color (pale yellow when seen in color) at pH7.3. FIG. 6B shows the absorption spectrum of this first modified CRAPIIpolypeptide (SEQ ID NO:43). Note that the first modified CRABPIIpolypeptide has two strong absorption maxima at pH 5.0, one at about 400nm and the other at about 610 nm. However, the absorption at about 610nm of this first CRABPII polypeptide is greatly reduced at pH 7.3. FIG.6C shows the absorption spectrum of a second modified CRAPII polypeptide(SEQ ID NO:44), which also has two strong absorption maxima at pH 5.0,one at about 375 nm and the other at about 600 nm However, theabsorption at about 600 nm of this second modified CRABPII polypeptide(SEQ ID NO:44) is greatly reduced at pH 7.3. FIG. 6D shows that thesecond modified CRABPII polypeptide (SEQ ID NO:44) has a darker color(purple when seen in color) at pH 5.0 and a lighter color (pale orangewhen seen in color) at pH 7.3. Thus, these modified CRABPII polypeptidesare colorimetric protein-based pH sensors capable of changing theirlight absorption and transmission properties in response to pH changesover a range of pH conditions spanning at least pH 5.0-7.5.

FIGS. 7A and B shows that modified CRABPII polypeptides are remarkablystable to acid. The absorption of a selected modified CRABPIIpolypeptide (SEQ ID NO:42) under various pH conditions in the presenceof retinal was measured. FIG. 7A shows that the polypeptide retainssecondary and tertiary structures contributing to the unique lightabsorption properties of the polypeptide over a wide range of pH values.Thus, the polypeptide exhibits extraordinary stability towardsacidification down to pH 1.6. FIG. 7B graphically illustrates the shiftin maximal wavelength of absorption with pH for this modified CRABPIIpolypeptide (SEQ ID NO:42).

FIG. 8 shows that modified CRABPII polypeptides are remarkably stable totemperature. The CD spectrum shows that while a CRABPII polypeptide withSEQ ID NO:45 unfolds at around 50° C., the modified CRABPII polypeptidewith SEQ ID NO:42 is stable up to 80° C. Increasing values along they-axis represent increased disorder in the secondary and/or tertiarystructures of the polypeptides. Thus, the modified CRABPII polypeptidesdescribed herein can be stabilized by introduction of specific aminoacid changes (e.g., selected from those in the modified CRABPIIpolypeptide with SEQ ID NO:42).

DETAILED DESCRIPTION OF THE INVENTION

The invention described herein relates to intracellular lipid bindingproteins (iLBPs) that are modified to absorb, emit, fluoresce and/ortransmit light in a variety of wavelengths when bound to a retinoid orfluorescent dye, and nucleic acids encoding such modified iLBPs. Suchmodified iLBPs are useful colorimetric and/or fluorescent labelingagents that can be fused to other molecules of interest. For example,the modified iLBPs of the invention can be used to label targetbiomolecules in vivo to permit observation and analysis of thebiomolecules' location, interactions and activities. Because thecolorimetric/fluorescent iLBP proteins of the invention are readilymodified to emit light at different wavelengths, several targetbiomolecules can be monitored at once by employing differentcolorimetric/fluorescent proteins.

In general, according to the invention, the wavelength of lighttransmitted or emitted depends upon the polarity of the iLBP pocket thatbinds a retinoid or other dye ligand. Thus, for example, increasednegative polarity in the pocket near a ring moiety of the retinoid ordye ligand and/or decreased negative polarity in the region of a Schiffbase formed between the iLBP and the dye ligand yields an iLBP:ligandcomplex that transmits light with a longer (more red) wavelength.Conversely, the light transmitted by an iLBP:ligand complex is moreblue-shifted (shorter wavelength) when the Schiff base region has morenegative polarity and the ring of the retinoid/dye ligand has decreasednegative polarity. The inventors have modulated the sequences of iLBPproteins to generate modified iLBP polypeptides that transmit or emitlight at a variety of wavelengths.

Intracellular Lipid Binding Proteins and Nucleic Acids

According to the invention, intracellular lipid binding proteins (iLBPs)can be modified to form fluorescent and colorimetric labeling agentsthat absorb and transmit light at diverse wavelengths when bound to aretinoid or fluorescent dye ligand. As illustrated herein, a wild typeintracellular lipid binding protein typically does not transmitsignificant light, especially when the wild type iLBP does not bind aretinoid or fluorescent dye ligand via a Schiff base.

iLBPs are low molecular mass proteins (14-16 kDa) that generally have acommon structural fold. The iLBP family likely arose through duplicationand diversification of an ancestral iLBP gene. Members of the family ofintracellular lipid binding proteins (iLBPs) can facilitate cytoplasmictransport of lipophilic ligands, such as long-chain fatty acids andretinoids. Thus, iLBPs naturally form a complex with long-chain fattyacids and retinoids. However, wild type iLBPs typically do not form aSchiff base linkage to the associated long-chain fatty acid or retinoidmolecule.

As illustrated herein, when an iLBP does bind a retinoid or dye ligandvia Schiff base formation, a stable iLBP:ligand complex forms thattransmits or emits light. By modulating the polarity of the retinoid/dyeligand binding pocket through substitution of one or more iLBP aminoacids, the wavelength as which the iLBP:ligand complex transmits oremits light can also be modulated.

Examples of iLBP proteins that can be used to generatecolorimetric/fluorescent protein:ligand complexes include the cellularretinoic acid binding protein II (CRABPII), cellular retinol bindingprotein II (CRBPII), liver-type fatty acid binding protein (L-FABP), theintestinal fatty acid binding protein (1-FABP), and the ileal lipidbinding protein (ilbp). In some embodiments, the iLBP selected forgenerating a fluorescent and colorimetric protein:ligand complex is aCellular Retinoic Acid Binding Protein II (CRABPII) and/or CellularRetinol Binding Protein II (CRBPII).

As used herein, a colorimetric and/o fluorescent protein or labelingagent is a member of the intracellular lipid binding protein (iLBP)family that has a modified amino acid sequence thereby generating whatis referred to as a modified iLBP polypeptide. Such a modified iLBPpolypeptide can transmit or emit light when bound to a retinoid orfluorescent dye molecule. In some embodiments, the iLBP polypeptide hasbeen modified so that an amino acid at any of positions 102-135 can forma Schiff base with a retinoid (e.g., retinal) or a fluorescent dyeligand. Also in some embodiments, the iLBP family member is CRABPII orCRBPII, which is modified to generate a colorimetric and/o fluorescentprotein or labeling agent (also referred to as a modified iLBPpolypeptide).

Examples of amino acid and nucleic acid sequences for different typesand species of iLBPs, including CRABPII and CRBPII polypeptides can befound in the art, for example, in the National Center for BiotechnologyInformation (NCBI) database. See website at ncbi.nlm.nih.gov. The aminoacid sequences for various iLBPs can have a methionine at theN-terminus. However, as is known to one of skill in the art, themethionine can be removed by post-translational processing, particularlyin eukaryotic cells. Therefore, in some embodiments the N-terminalmethionine is removed or is not present on the polypeptide sequencesdescribed and claimed herein.

One sequence for a wild type human cellular retinol binding protein II(hCRBPII) polypeptide is provided by the NCBI database as accessionnumber P50120.3 (GI:62297500), which is readily used as a basis forgenerating iLBP fluorescent and colorimetric labeling agents. Thesequence for this P50120.3 (GI:62297500) polypeptide is provided belowfor easy reference as SEQ ID NO:1.

  1 MTRDQNGTWE MESNENFEGY MKALDIDFAT RKIAVRLTQT  41KVIDQDGDNF KTKTTSTFRN YDVDFTVGVE FDEYTKSLDN  81RHVKALVTWE GDVLVCVQKG EKENRGWKQW IEGDKLYLEL 121 TCGDQVCRQV FKKK

A nucleic acid sequence for this wild type human cellular retinolbinding protein II polypeptide is available in the NCBI database asaccession number NM_(—)004164.2 (GI:40354213). This sequence is providedbelow for easy reference as SEQ ID NO:2.

  1 CCTGCTCCTT GCCATCCACC ACAAACCCTC ACCGAACCAG  41TGGCCACCAC CATGACAAGG GACCAGAATG GAACCTGGGA  81GATGGAGAGT AATGAAAACT TTGAGGGCTA CATGAAGGCC 121CTGGATATTG ATTTTGCCAC CCGCAAGATT GCAGTACGTC 161TCACTCAGAC GAAGGTTATT GATCAAGATG GTGATAACTT 201CAAGACAAAA ACCACTAGCA CATTCCGCAA CTATGATGTG 241GATTTCACTG TTGGAGTAGA GTTTGACGAG TACACAAAGA 281GCCTGGATAA CCGGCATGTT AAGGCACTGG TCACCTGGGA 321AGGTGATGTC CTTGTGTGTG TGCAAAAGGG GGAGAAGGAG 361AACCGCGGCT GGAAGCAGTG GATTGAGGGG GACAAGCTGT 401ACCTGGAGCT GACCTGTGGT GACCAGGTGT GCCGTCAAGT 441GTTCAAAAAG AAATGATGGC GACGTGGGAG GCCTGCCAAG 481CACAAGCTCC CCACTGCCCA CACTGAGTGG TCTACTGGCT 521TTGAGAAACA GCTGTGGGGA CCTTCCCACT CTTGACAGAG 561CCCCATTAAG GCATCTGGGT GGGTTTTAAA CAGAATGCCT 601ATGTAGCAGT GATAGACATA TTCCCCTCCT TTGAAACCTA 641GCATTAAATG GAAAAACAAA AATTACTCCC ATATTTTGAA 681 ACCCTTTAAA AAAAAAAAAAThis wild type hCRBPII nucleic acid, as well as other wild type iLBPnucleic acids, are useful for making modified nucleic acids that encodemodified iLBP polypeptides with useful light absorption and transmissionproperties. Thus, a selected wild type iLBP nucleic acid can be modifiedby procedures available to those of skill in the art to encode modifiediLBP polypeptides. Recombinant expression of the encoded modified iLBPsnot only yields useful quantities of colorimetric/fluorescent iLBPpolypeptides but also can be used for in vivo analysis of biologicalprocesses and biological products, as described in more detail below.

Other CRBPII sequences in addition to the SEQ ID NO:1 sequence can beused as a basis for generating modified iLBPs that are useful asfluorescent and colorimetric labeling agents. Thus, another human CRBPIIpolypeptide sequence that is available in the NCBI database as accessionnumber AAC50162.1 (GI:535390). This sequence is provided below for easyreference as SEQ ID NO:3.

  1 MTRDQNGTWE MESNENFEGY MKALDIDFAT PKIAVRLTQT  41KVIDQDGDNF KTKTTSTFRN YDVDFTVGVE FDEYTKSLDN  81RHVKALVTWE GDVLVCVQKG EKENRGWKQW IEGDKLYLEL 121 TCGDQVCRQV FKKK

A nucleic acid sequence for this human cellular retinol binding proteinII polypeptide is available in the NCBI database as accession numberNM_(—)004164.2 (GI:40354213). This sequence is provided below for easyreference as SEQ ID NO:4.

  1 CCTGCTCCTT GCCATCCACC ACAAACCCTC ACCGAACCAG  41TGGCCACCAC CATGACAAGG GACCAGAATG GAACCTGGGA  81GATGGAGAGT AATGAAAACT TTGAGGGCTA CATGAAGGCC 121CTGGATATTG ATTTTGCCAC CCGCAAGATT GCAGTACGTC 161TCACTCAGAC GAAGGTTATT GATCAAGATG GTGATAACTT 201CAAGACAAAA ACCACTAGCA CATTCCGCAA CTATGATGTG 241GATTTCACTG TTGGAGTAGA GTTTGACGAG TACACAAAGA 281GCCTGGATAA CCGGCATGTT AAGGCACTGG TCACCTGGGA 321AGGTGATGTC CTTGTGTGTG TGCAAAAGGG GGAGAAGGAG 361AACCGCGGCT GGAAGCAGTG GATTGAGGGG GACAAGCTGT 401ACCTGGAGCT GACCTGTGGT GACCAGGTGT GCCGTCAAGT 441GTTCAAAAAG AAATGATGGC GACGTGGGAG GCCTGCCAAG 481CACAAGCTCC CCACTGCCCA CACTGAGTGG TCTACTGGCT 521TTGAGAAACA GCTGTGGGGA CCTTCCCACT CTTGACAGAG 561CCCCATTAAG GCATCTGGGT GGGTTTTAAA CAGAATGCCT 601ATGTAGCAGT GATAGACATA TTCCCCTCCT TTGAAACCTA 641GCATTAAATG GAAAAACAAA AATTACTCCC ATATTTTGAA 681 ACCCTTTAAA AAAAAAAAAA

In some cases the modified polypeptides of the invention have amethionine at their N-terminus, but in other cases the methionine is notpresent. For example, when the methionine is removed from the N-terminusof the SEQ ID NO:1 hCRBPII polypeptide, this polypeptide has thefollowing sequence (SEQ ID NO:5).

  1 TRDQNGTWEM ESNENFEGYM KALDIDFATR KIAVRLTQTK  41VIDQDGDNFK TKTTSTFRNY DVDFTVGVEF DEYTKSLDNR  81HVKALVTWEG DVLVCVQKGE KENRGWKQWI EGDKLYLELT 121 CGDQVCRQVF KKK

As illustrated herein, the light absorption and transmission propertiesof such an hCRBPII polypeptide can be modulated by modulating thesequence of hCRBPII polypeptide. This can be done by proceduresavailable in the art, for example, by recombinant manipulation orsite-directed mutagenesis of a nucleic acid encoding the hCRBPII. Thus,for example, when a glutamine (Q) amino acid at about positions 107-109(preferably position 108) is replaced with a lysine (K) amino acid, amodified hCRBPII polypeptide is generated with somewhat differentphysical and chemical properties, in addition to somewhat differentlight absorption/transmission properties. One example of such a modifiedCRBPII polypeptide is called a Q108K hCRBPII polypeptide, which can havethe following sequence (SEQ ID NO: 6).

  1 TRDQNGTWEM ESNENFEGYM KALDIDFATR KIAVRLTQTK  41VIDQDGDNFK TKTTSTFRNY DVDFTVGVEF DEYTKSLDNR  81HVKALVTWEG DVLVCVQKGE KENRGWK K WI EGDKLYLELT 121 CGDQVCRQVF KKKNote that the Q108K nomenclature means that while a glutamine (Q) atabout position 108 is present in the wild type protein that glutaminehas been replaced by a lysine (K) in the Q108K hCRBPII polypeptideidentified as SEQ ID NO:6.

Such a Q108K hCRBPII polypeptide maximally absorbs light at 506 nm andadopts a favorable three-dimensional structure for positioning thelysine at position 108 to attack the retinal aldehyde to form aprotonated Schiff base. However, the folding of this protein brings alysine residue at about position 39-41 close to the Schiff base thatforms between the retinal aldehyde and the nitrogen of the lysine atposition 108. This lysine at position 39-41 perturbs the pK_(a) of theprotonated Schiff base, which affects the light absorption/transmissionproperties of the polypeptide as well as the stability of the complexformed between retinal and the Q108K hCRBPII polypeptide. To restore thepKa a counter ion can be introduced or the lysine at position 39-41 canbe replaced with a less charged amino acid.

In some embodiments, the lysine at position 39-41 is replaced with aleucine. For example, when the lysine (K) at position 40 of the Q108KhCRBPII polypeptide is replaced with a leucine (L) amino acid, amodified Q108K; K40L hCRBPII polypeptide is formed, which has thefollowing sequence (SEQ ID NO: 7).

  1 TRDQNGTWEM ESNENFEGYM KALDIDFATR KIAVRLTQT L  41VIDQDGDNFK TKTTSTFRNY DVDFTVGVEF DEYTKSLDNR  81HVKALVTWEG DVLVCVQKGE KENRGWK K WI EGDKLYLELT 121 CGDQVCRQVF KKKThe wavelength at which the Q108K; K40L hCRBPII polypeptide incombination with retinal maximally absorbs light is 508 nm.

Another modified hCRBPII polypeptide with not only the Q108Ksubstitution but also a replacement of threonine (T) with aspartic acid(D) at position 50-52 (e.g., position 51) also has useful lightabsorption and transmission properties. The sequence of this Q108K; T51DhCRBPII polypeptide is shown below (SEQ ID NO:8).

  1 TRDQNGTWEM ESNENFEGYM KALDIDFATR KIAVRLTQTK  41 VIDQDGDNFK  DKTTSTFRNY DVDFTVGVEF DEYTKSLDNR  81 HVKALVTWEG DVLVCVQKGE KENRGWK KWI EGDKLYLELT 121 CGDQVCRQVF KKKThe wavelength at which the Q108K; T51D hCRBPII polypeptide incombination with retinal maximally absorbs light is 474 nm.

Studies indicate that the Q108K; K40L hCRBPII polypeptide is more stablethan the Q108K; T51D hCRBPII polypeptide. Hence, in some embodimentsQ108K; K40L hCRBPII polypeptides are used a platform for generatingother modified iLBP colorimetric/fluorescent proteins.

To generate a variety of fluorescent and colorimetric labeling agentsthat absorb and transmit light at a variety of different wavelengths thehCRBPII polypeptide (e.g. the Q108K; K40L hCRBPII polypeptide) sequencecan be altered in a variety of ways.

For example, the tyrosine at any of positions 59-61 can be changed to atryptophan. When this is done at position 60 of the Q108K; K40L hCRBPIIpolypeptide, a polypeptide that maximally absorbs light at 512 nm isgenerated that is called the Q108K; K40L; Y60W hCRBPII polypeptide, withthe following sequence (SEQ ID NO:9).

  1 TRDQNGTWEM ESNENFEGYM KALDIDFATR KIAVRLTQT L  41VIDQDGDNFK TKTTSTFRN W  DVDFTVGVEF DEYTKSLDNR  81HVKALVTWEG DVLVCVQKGE KENRGWK K WI EGDKLYLELT 121 CGDQVCRQVF KKK

In another example, the threonine at any of positions 50-52 (e.g.,position 51) can be replaced with a valine. For example, if thethreonine (T) at position 51 of the Q108K; K40L hCRBPII polypeptide isreplaced with a valine (V), the resulting Q108K; K40L; T51V hCRBPIIpolypeptide has the following sequence (SEQ ID NO:10).

  1 TRDQNGTWEM ESNENFEGYM KALDIDFATR KIAVRLTQT L  41 VIDQDGDNFK  VKTTSTFRNY DVDFTVGVEF DEYTKSLDNR  81 HVKALVTWEG DVLVCVQKGE KENRGWK KWI EGDKLYLELT 121 CGDQVCRQVF KKKThe wavelength at which the Q108K; K40L; T51V hCRBPII polypeptide, incombination with retinal, maximally absorbs light is 533 nm.

A replacement of the arginine at any of positions 57-59 with anotheramino acid can also modulate the wavelength at which an hCRBPIIpolypeptide absorbs and/or transmits light. For example, if the arginine(R) at position 58 of the Q108K; K40L hCRBPII polypeptide is replacedwith a phenylalanine (F), the resulting Q108K; K40L; R58F hCRBPIIpolypeptide maximally absorbs light at 524 nm, and has the followingsequence (SEQ ID NO:11).

  1 TRDQNGTWEM ESNENFEGYM KALDIDFATR KIAVRLTQT L  41 VIDQDGDNFK TKTTSTFF NY DVDFTVGVEF DEYTKSLDNR  81 HVKALVTWEG DVLVCVQKGE KENRGWK KWI EGDKLYLELT 121 CGDQVCRQVF KKK

But if the arginine (R) at position 58 of the Q108K; K40L hCRBPIIpolypeptide is replaced with a tyrosine (Y), the resulting Q108K; K40L;R58Y hCRBPII polypeptide maximally absorbs light at 535 nm, and has thefollowing sequence (SEQ ID NO:12).

  1 TRDQNGTWEM ESNENFEGYM KALDIDFATR KIAVRLTQT L  41 VIDQDGDNFK TKTTSTFY NY DVDFTVGVEF DEYTKSLDNR  81 HVKALVTWEG DVLVCVQKGE KENRGWK KWI EGDKLYLELT 121 CGDQVCRQVF KKK

Still further modulation of the light absorption and transmissionproperties of CRBPII polypeptides can be achieved by making severalamino acid replacements at once. Thus, for example, if the arginine (R)at position 58 of a Q108K; K40L; T51V hCRBPII polypeptide is replacedwith a tyrosine (Y) the resulting Q108K; K40L; T51V; R58Y hCRBPIIpolypeptide maximally absorbs light is 563 nm as opposed to 533 nm forthe Q108K; K40L; T51V hCRBPII or at 524 nm for the Q108K; K40L; R58FhCRBPII polypeptide. The sequence of the Q108K; K40L; T51V; R58Y hCRBPIIpolypeptide is as follows (SEQ ID NO:13).

  1 TRDQNGTWEM ESNENFEGYM KALDIDFATR KIAVRLTQT L  41 VIDQDGDNFK  VKTTSTF Y NY DVDFTVGVEF DEYTKSLDNR  81 HVKALVTWEG DVLVCVQKGE KENRGWK KWI EGDKLYLELT 121 CGDQVCRQVF KKK

Changing the arginine (R) at position 58 of a Q108K; K40L; T51V hCRBPIIpolypeptide to a tryptophan (W) results in a Q108K; K40L; T51V; R58WhCRBPII polypeptide with the following sequence (SEQ ID NO:14).

  1 TRDQNGTWEM ESNENFEGYM KALDIDFATR KIAVRLTQT L  41 VIDQDGDNFK  VKTTSTF W NY DVDFTVGVEF DEYTKSLDNR  81 HVKALVTWEG DVLVCVQKGE KENRGWK KWI EGDKLYLELT 121 CGDQVCRQVF KKK

The light absorption and transmission properties of CRBPII polypeptidescan also be modulated by replacement of a threonine at any of positions52-54. For example, replacement of the threonine at position 53 of theQ108K; K40L; T51V; R58W hCRBPII polypeptide yields a polypeptide thatmaximally absorbs light at 585 nm that is referred as the Q108K; K40L;T51V; R58W; T53C hCRBPII polypeptide, which has the following sequence(SEQ ID NO:15).

  1 TRDQNGTWEM ESNENFEGYM KALDIDFATR KIAVRLTQT L  41 VIDQDGDNFK  V K CTSTF W NY DVDFTVGVEF DEYTKSLDNR  81 HVKALVTWEG DVLVCVQKGE KENRGWK KWI EGDKLYLELT 121 CGDQVCRQVF KKK

Replacement of a threonine at any of positions 28-30 with another aminoacid also modulates the light absorption and transmission properties.For example, replacing a threonine (T) at position 29 of the Q108K;K40L; T51V; R58W; T53C hCRBPII polypeptide with a leucine, yields apolypeptide with the following sequence (SEQ ID NO:16), that is referredto as the Q108K; K40L; T51V; R58W; T53C; T29L hCRBPII polypeptide.

  1 TRDQNGTWEM ESNENFEGYM KALDIDFA L R KIAVRLTQT L  41 VIDQDGDNFK  V K CTSTF W NY DVDFTVGVEF DEYTKSLDNR  81 HVKALVTWEG DVLVCVQKGE KENRGWK KWI EGDKLYLELT 121 CGDQVCRQVF KKK

Replacement of a tyrosine at any of positions 18-20 can also modulatethe light absorption and transmission properties. For example, when atyrosine at position 19 of the Q108K; K40L; T51V; R58W; T53C; T29LhCRBPII polypeptide is replaced with a tryptophan, a polypeptide with alight absorption maximum of 591 is generated, which is referred to asthe Q108K; K40L; T51V; R58W; T53C; T29L; Y19W hCRBPII polypeptide. Thispolypeptide has the following sequence (SEQ ID NO:17).

  1 TRDQNGTWEM ESNENFEG W M KALDIDFA L R KIAVRLTQT L  41 VIDQDGDNFK  V KC TSTF W NY DVDFTVGVEF DEYTKSLDNR  81 HVKALVTWEG DVLVCVQKGE KENRGWK KWI EGDKLYLELT 121 CGDQVCRQVF KKK

Replacement of a glutamine (Q) amino acid at any of positions 3-5 canalso modulate the light absorption and transmission properties. Forexample, when a glutamine at position 4 of the Q108K; K40L; T51V; R58W;T53C; T29L; Y19W hCRBPII polypeptide is changed to a arginine (R), apolypeptide with a light absorption maximum of 622 nm is generated,which is referred to as the Q108K; K40L; T51V; R58W; T53C; T29L; Y19W;Q4R hCRBPII polypeptide. This polypeptide has the following sequence(SEQ ID NO:18).

  1 TRD R NGTWEM ESNENFEG W M KALDIDFA L R KIAVRLTQT L  41 VIDQDGDNFK  VK C TSTF W NY DVDFTVGVEF DEYTKSLDNR  81 HVKALVTWEG DVLVCVQKGE KENRGWK KWI EGDKLYLELT 121 CGDQVCRQVF KKK

The following table summarizes the light absorption/transmissionproperties of various CRBPII polypeptides that are complexed withretinal.

TABLE 1 Maximum Absorption Wavelength, Kd/nM and pKa Values for ModifiedCRBPII polypeptides Modified CRBPII λ_(max) (nm) K_(d)/nM pK_(a) Q108K506 48 ± 4  <6.0 Q108K; T51D 474 67 ± 6  9.2 Q108K; K40L 508 29 ± 5  7.9Q108K; K40L; Y60W 512 4 ± 8 7.1 Q108K; K40L; T51V 533 19 ± 7  8.3 Q108K;K40L; R58F 524 27 ± 6  8.6 Q108K; K40L; R58Y 535 10 ± 7  9.5 Q108K;K40L; T51V; R58Y 563 40 ± 5  10.1 Q108K; K40L; T51V; R58Y; Y19W 565 47 ±5  10.3 Q108K; K40L; T51V; R58W; T53C; 591 38 ± 10 8.2 T29L; Y19W Q108K;K40L; T51V; R58W; T53C; 622 183 ± 11  6.5 T29L; Y19W; Q4RAs illustrated by the data in Table 1, a large increase in the pKa valueof the CRBPII polypeptide is observed when the arginine at any ofpositions 57-59 is replaced with another amino acid (e.g., at position58, R58Y), even though the amino acid at position 58 is distant from thelocus of Schiff base formation.

Moreover, the type of amino acid selected for replacement alters thelight absorption and transmission properties of the polypeptide:retinoidcomplex. For example, when a variety of different amino acids are usedinstead of a glutamine at any of positions 3-5, the resulting CRBPIIpolypeptide absorbs/transmits light at a variety of differentwavelengths.

As indicated above, when the glutamine at position 4 of the Q108K; K40L;T51V; R58W; T53C; T29L; Y19W hCRBPII polypeptide is changed to anarginine (R) a polypeptide with a light absorption maximum of 622 nm isgenerated, which is referred to as the Q108K; K40L; T51V; R58W; T53C;T29L; Y19W; Q4R hCRBPII polypeptide. This polypeptide has the followingsequence (SEQ ID NO:19).

  1 TRD R NGTWEM ESNENFEG W M KALDIDFA L R KIAVRLTQT L  41 VIDQDGDNFK  VK C TSTF W NY DVDFTVGVEF DEYTKSLDNR  81 HVKALVTWEG DVLVCVQKGE KENRGWK KWI EGDKLYLELT 121 CGDQVCRQVF KKK

However, when the glutamine at position 4 of the Q108K; K40L; T51V;R58W; T53C; T29L; Y19W hCRBPII polypeptide is changed to an tryptophan(W) a polypeptide with a light absorption maximum of 613 nm isgenerated, which is referred to as the Q108K; K40L; T51V; R58W; T53C;T29L; Y19W; Q4W hCRBPII polypeptide. This polypeptide has the followingsequence (SEQ ID NO:20).

  1 TRD W NGTWEM ESNENFEG W M KALDIDFA L R KIAVRLTQT L  41 VIDQDGDNFK  VK C TSTF W NY DVDFTVGVEF DEYTKSLDNR  81 HVKALVTWEG DVLVCVQKGE KENRGWK KWI EGDKLYLELT 121 CGDQVCRQVF KKK

When the glutamine at position 4 of the Q108K; K40L; T51V; R58W; T53C;T29L; Y19W hCRBPII polypeptide is changed to an asparagine (N) apolypeptide with the same light absorption maximum of 613 nm isgenerated, which is referred to as the Q108K; K40L; T51V; R58W; T53C;T29L; Y19W; Q4N hCRBPII polypeptide. This polypeptide has the followingsequence (SEQ ID NO:21).

  1 TRD N NGTWEM ESNENFEG W M KALDIDFA L R KIAVRLTQT L  41 VIDQDGDNFK  VK C TSTF W NY DVDFTVGVEF DEYTKSLDNR  81 HVKALVTWEG DVLVCVQKGE KENRGWK KWI EGDKLYLELT 121 CGDQVCRQVF KKK

Use of a threonine (T) at the position of the glutamine at position 4 ofthe Q108K; K40L; T51V; R58W; T53C; T29L; Y19W hCRBPII polypeptide yieldsa polypeptide with a light absorption maximum of 610 nm, which isreferred to as the Q108K; K40L; T51V; R58W; T53C; T29L; Y19W; Q4ThCRBPII polypeptide. This polypeptide has the following sequence (SEQ IDNO:22).

  1 TRD T NGTWEM ESNENFEG W M KALDIDFA L R KIAVRLTQT L  41 VIDQDGDNFK  VK C TSTF W NY DVDFTVGVEF DEYTKSLDNR  81 HVKALVTWEG DVLVCVQKGE KENRGWK KWI EGDKLYLELT 121 CGDQVCRQVF KKK

Use of a glutamic acid (E) at the position of the glutamine at position4 of the Q108K; K40L; T51V; R58W; T53C; T29L; Y19W hCRBPII polypeptideyields a polypeptide with a light absorption maximum of 590 nm, which isreferred to as the Q108K; K40L; T51V; R58W; T53C; T29L; Y19W; Q4EhCRBPII polypeptide. This polypeptide has the following sequence (SEQ IDNO:23).

  1 TRD E NGTWEM ESNENFEG W M KALDIDFA L R KIAVRLTQT L  41 VIDQDGDNFK  VK C TSTF W NY DVDFTVGVEF DEYTKSLDNR  81 HVKALVTWEG DVLVCVQKGE KENRGWK KWI EGDKLYLELT 121 CGDQVCRQVF KKK

Use of a histidine (H) at the position of the glutamine at position 4 ofthe Q108K; K40L; T51V; R58W; T53C; T29L; Y19W hCRBPII polypeptide yieldsa polypeptide with a light absorption maximum of 585 nm, which isreferred to as the Q108K; K40L; T51V; R58W; T53C; T29L; Y19W; Q4HhCRBPII polypeptide. This polypeptide has the following sequence (SEQ IDNO:24).

  1 TRD H NGTWEM ESNENFEG W M KALDIDFA L R KIAVRLTQT L  41 VIDQDGDNFK  VK C TSTF W NY DVDFTVGVEF DEYTKSLDNR  81 HVKALVTWEG DVLVCVQKGE KENRGWK KWI EGDKLYLELT 121 CGDQVCRQVF KKK

Use of a lysine (K) at the position of the glutamine at position 4 ofthe Q108K; K40L; T51V; R58W; T53C; T29L; Y19W hCRBPII polypeptide yieldsa polypeptide with a light absorption maximum of 616 nm, which isreferred to as the Q108K; K40L; T51V; R58W; T53C; T29L; Y19W; Q4KhCRBPII polypeptide. This polypeptide has the following sequence (SEQ IDNO:25).

  1 TRD K NGTWEM ESNENFEG W M KALDIDFA L R KIAVRLTQT L  41 VIDQDGDNFK  VK C TSTF W NY DVDFTVGVEF DEYTKSLDNR  81 HVKALVTWEG DVLVCVQKGE KENRGWK KWI EGDKLYLELT 121 CGDQVCRQVF KKK

Use of a lysine (K) at the position of the glutamine at position 4 ofthe Q108K; K40L; T51V; R58W; T53C; T29L; Y19W hCRBPII polypeptide yieldsa polypeptide with a light absorption maximum of 614 nm, which isreferred to as the Q108K; K40L; T51V; R58W; T53C; T29L; Y19W; Q4LhCRBPII polypeptide. This polypeptide has the following sequence (SEQ IDNO:26).

  1 TRD L NGTWEM ESNENFEG W M KALDIDFA L R KIAVRLTQT L  41 VIDQDGDNFK  VK C TSTF W NY DVDFTVGVEF DEYTKSLDNR  81 HVKALVTWEG DVLVCVQKGE KENRGWK KWI EGDKLYLELT 121 CGDQVCRQVF KKK

Use of a phenylalanine (F) at the position of the glutamine at position4 of the Q108K; K40L; T51V; R58W; T53C; T29L; Y19W hCRBPII polypeptideyields a polypeptide with a light absorption maximum of 613 nm, which isreferred to as the Q108K; K40L; T51V; R58W; T53C; T29L; Y19W; Q4FhCRBPII polypeptide. This polypeptide has the following sequence (SEQ IDNO:27).

  1 TRD F NGTWEM ESNENFEG W M KALDIDFA L R KIAVRLTQT L  41 VIDQDGDNFK  VK C TSTF W NY DVDFTVGVEF DEYTKSLDNR  81 HVKALVTWEG DVLVCVQKGE KENRGWK KWI EGDKLYLELT 121 CGDQVCRQVF KKK

The following table summarizes the light absorption/transmissionproperties of various Q108K; K40L; T51V; R58W; T53C; T29L; Y19W CRBPIIpolypeptides where the glutamine at position 4 is replaced with avariety of different amino acids and the resulting polypeptide iscomplexed with retinal.

TABLE 2 Maximum Absorption Wavelength, Kd/nM and pKa Values for Q108K;K40L; T51V; R58W; T53C; T29L; Y19W; Q4 CRBPII polypeptides ModifiedCRBPII λ_(max) (nm) K_(d)/nM pK_(a) Q108K; K40L; T51V; R58W; T53C; 59138 ± 10 8.2 T29L; Y19W; Q4 (no replacement of the glutamine at position4) Q108K; K40L; T51V; R58W; T53C; 613 103 ± 10  7.7 T29L; Y19W; Q4WQ108L; K40L; T51V; R58W; T53C; 614 57 ± 8  7.9 T29L; Y19W; Q4F Q108K;K40L; T51V; R58W; T53C; 613 58 ± 12 7.5 T29L; Y19W; Q4L Q108K; K40L;T51V; R58W; T53C; 613 65 ± 12 7.2 T29L; Y19W; Q4N Q108K; K40L; T51V;R58W; T53C; 610 63 ± 8  7.8 T29L; Y19W; Q4T Q108K; K40L; T51V; R58W;T53C; 590 162 ± 20  ND T29L; Y19W; Q4E Q108K; K40L; T51V; R58W; T53C;585 18 ± 5  7.9 T29L; Y19W; Q4H Q108K; K40L; T51V; R58W; T53C; 616 12 ±8  7.2 T29L; Y19W; Q4K Q108K; K40L; T51V; R58W; T53C; 622 183 ± 11  6.5T29L; Y19W; Q4R

Thus, modulating not only the position of the amino acid replacement,but also the type of amino acid placed in a position modulates the lightabsorption and transmission properties of a CRBPII polypeptide:retinoidcomplex. In the example above, the glutamine at position 4 of the CRBPIIpolypeptide is about 4.5 Å away from the Schiff base formed between thepolypeptide and retinal. As shown in Table 2, replacement of thisglutamine at position 4 has a large effect on the wavelength of lightabsorbed and transmitted as well as a significant effect upon the pKa ofthe protonated Schiff base (PSB) formed between retinal and the lysine(or glutamine) at position 108. Removal of the glutamine at position 4destabilizes the ground state of the protonated Schiff base, resultingin a lower pKa and a more red-shifted CRBPII:retinal complex. Placementof a positive charge at position 4 (e.g., with arginine) generates avery red-shifted CRBPII:retinal complex, but this complex also has a lowpKa.

Replacement of a threonine at any of positions 32-34 with another aminoacid also modulates the light absorption and transmission properties.For example, replacing an alanine (A) at position 33 of the Q108K; K40L;T51V; R58W; T53C; T29L; Y19W; Q4R hCRBPII polypeptide with a tryptophan,yields a polypeptide with the following sequence (SEQ ID NO: 28), thatis referred to as the Q108K; K40L; T51V; R58W; T53C; T29L; Y19W; Q4R;A33W hCRBPII polypeptide. This polypeptide is an even more red-shiftedCRBPII; retinal complex, with a maximal wavelength of absorption at 644nm.

  1 TRD R NGTWEM ESNENFEG W M KALDIDFA L R KI W VRLTQT L  41 VIDQDGDNFK V K C TSTF W NY DVDFTVGVEF DEYTKSLDNR  81 HVKALVTWEG DVLVCVQKGE KENRGWKK WI EGDKLYLELT 121 CGDQVCRQVF KKK

As is known to the skilled artisan, sequence variation can occur acrossspecies. Thus, a rat cellular retinol binding protein II polypeptidesequence with an NCBI accession number of P06768.3 (GI:132399) has aslightly different sequence than the human cellular retinol bindingprotein II polypeptide sequences. This rat sequence is provided belowfor easy reference as SEQ ID NO:29.

  1 TKDQNGTWEM ESNENFEGYM KALDIDFATR KIAVRLTQTK 41 IIVQDGDNFK TKTNSTFRNY DLDFTVGVEF DEHTKGLDGR 81 NVKTLVTWEG NTLVCVQKGE KENRGWKQWV EGDKLYLELT 121 CGDQVCRQV FKKK

A nucleic acid sequence for this rat cellular retinol binding protein IIpolypeptide is available in the NCBI database as accession numberNM_(—)012640.2 (GI:78126162). This sequence is provided below for easyreference as SEQ ID NO:30.

  1 GCAGCTTGTT CCTTCACGGT CACCAAACGT CCGCATCAAA 41 CCAGAGGCCG CCATCATGAC GAAGGACCAG AATGGAACCT 81 GGGAAATGGA GAGTAATGAG AACTTTGAAG GCTACATGAA121 GGCCCTAGAT ATTGATTTTG CCACCCGCAA GATTGCAGTG161 CGTCTGACTC AGACGAAGAT CATCGTTCAA GACGGTGATA201 ACTTCAAGAC AAAAACCAAC AGCACGTTCC GCAACTATGA241 CCTAGATTTC ACAGTGGGGG TGGAGTTTGA CGAACACACA281 AAGGGTCTGG ATGGCCGGAA CGTCAAGACC CTAGTCACCT321 GGGAAGGAAA CACCCTGGTG TGTGTGCAGA AAGGGGAGAA361 GGAGAATCGT GGCTGGAAGC AGTGGGTCGA GGGAGACAAG401 CTGTACCTGG AGCTGACCTG CGGTGACCAG GTGTGTCGAC441 AAGTGTTCAA AAAGAAGTGA TGGGCCCAGG GGAAGCCTGG481 AACATGTGTA GAGTTCTCTG CCATTCTGAA AAGCAGCATT521 GGGACTCCCT GGTTCCTGAC AGAGCCCCCC TTGCATCACC561 TGCCTGGGTT TGAAACAGGG TGTGTTAAAG GAACCTACCC601 CCTCCCCCTT AGAACCTATT ATTAAATAAA AAAACAAAAC641 ATCCTCTCGG CCTTTGAAAA AAAAAAAAAA AAAA

A mouse cellular retinol binding protein II polypeptide sequence isavailable in the NCBI database as accession number Q08652.2 (GI:730494).This sequence is provided below for easy reference as SEQ ID NO:31.

  1 TKDQNGTWEM ESNENFEGYM KALDIDFATR KIAVRLTQTK 41 IITQDGDNFK TKTNSTFRNY DLDFTVGVEF DEHTKGLDGR 81 HVKTLVTWEG NTLVCVQKGE KENRGWKQWV EGDKLYLELT 121 CGDQVCRQV FKKK

A nucleic acid sequence for this mouse cellular retinol binding proteinII polypeptide is available in the NCBI database as accession numberNM_(—)009034.4 (GI:255759937). This sequence is provided below for easyreference as SEQ ID NO:32.

  1 ATTTAGCATA GTCTCCCTGC AGCCTGTTCC TTCACAGTCA 41 CCGAACGTCC ACATCAAACC AGAGGCCACC ATCATGACGA 81 AGGACCAAAA TGGAACCTGG GAAATGGAGA GTAATGAGAA121 CTTTGAAGGC TACATGAAGG CCCTAGATAT TGATTTTGCC161 ACCCGCAAGA TCGCAGTGCG TCTGACTCAG ACGAAGATCA201 TCACTCAAGA CGGTGATAAC TTCAAGACGA AAACCAACAG241 CACGTTCCGC AACTACGACC TGGATTTCAC CGTCGGGGTG281 GAGTTTGACG AACACACAAA GGGCCTGGAC GGCCGACATG321 TCAAGACCCT GGTCACCTGG GAAGGCAACA CCCTCGTGTG361 TGTGCAGAAA GGGGAGAAGG AGAACCGTGG CTGGAAGCAG401 TGGGTGGAGG GAGACAAGCT GTACCTGGAG CTGACCTGCG441 GCGACCAGGT GTGCCGACAA GTGTTCAAAA AGAAGTGATG481 GGCACGGGAA AGCCTGGAAC ATGTGCAGAG TTCTCTGCCA521 GTTCCCCAAA GCAGCATGGG GACTCCTCCC ATTCCTGACA561 GAGCCCCCTT ACATCATCTG CCTGGGTTTA AACTGGAGTG601 TATAAAAGGA ACCTACCCCC CTCCCAGCCC CCCCCCCCAA641 GCTTGTTATT AAAGAAACAA AATGTCCTCT CA

Other types of polypeptides, which bind vitamin A-like molecules can beused for making fluorescent and colorimetric labeling agents. Forexample, cellular retinoic acid-binding protein 2 polypeptides (CRABPII)can be used for making fluorescent and colorimetric labeling agents.

One sequence for a human cellular retinoic acid-binding protein 2 aminoacid sequence (hCRABPII) polypeptide is provided in the NCBI database asaccession number NP_(—)001186652.1 (GI:315013542). This sequence isprovided below for easy reference as SEQ ID NO:33.

  1 MPNFSGNWKI IRSENFEELL KVLGVNVMLR KIAVAAASKP 41 AVEIKQEGDT FYIKTSTTVR TTEINFKVGE EFEEQTVDGR 81 PCKSLVKWES ENKMVCEQKL LKGEGPKTSW TRELTNDGEL 121 ILTMTADDVV CTRVYVRE

A nucleic acid sequence for this human cellular retinoic acid-bindingprotein 2 polypeptide is available in the NCBI database as accessionnumber NM_(—)001199723.1 (GI:315013541). This sequence is provided belowfor easy reference as SEQ ID NO:34.

   1 GATTCAAGTG CTGGCTTTGC GTCCGCTTCC CCATCCACTT  41 ACTAGCGCAG GAGAAGGCTA TCTCGGTCCC CAGAGAAGCC  81 TGGACCCACA CGCGGGCTAG ATCCAGAGAA CCTGACGACC 121 CGGCGACGGC GACGTCTCTT TTGACTAAAA GACAGTGTCC 161 AGTGCTCCAG CCTAGGAGTC TACGGGGACC GCCTCCCGCG 201 CCGCCACCAT GCCCAACTTC TCTGGCAACT GGAAAATCAT 241 CCGATCGGAA AACTTCGAGG AATTGCTCAA AGTGCTGGGG 281 GTGAATGTGA TGCTGAGGAA GATTGCTGTG GCTGCAGCGT 321 CCAAGCCAGC AGTGGAGATC AAACAGGAGG GAGACACTTT 361 CTACATCAAA ACCTCCACCA CCGTGCGCAC CACAGAGATT 401 AACTTCAAGG TTGGGGAGGA GTTTGAGGAG CAGACTGTGG 441 ATGGGAGGCC CTGTAAGAGC CTGGTGAAAT GGGAGAGTGA 481 GAATAAAATG GTCTGTGAGC AGAAGCTCCT GAAGGGAGAG 521 GGCCCCAAGA CCTCGTGGAC CAGAGAACTG ACCAACGATG 561 GGGAACTGAT CCTGACCATG ACGGCGGATG ACGTTGTGTG 601 CACCAGGGTC TACGTCCGAG AGTGAGTGGC CACAGGTAGA 641 ACCGCGGCCG AAGCCCACCA CTGGCCATGC TCACCGCCCT 681 GCTTCACTGC CCCCTCCGTC CCACCCCCTC CTTCTAGGAT 721 AGCGCTCCCC TTACCCCAGT CACTTCTGGG GGTCACTGGG 761 ATGCCTCTTG CAGGGTCTTG CTTTCTTTGA CCTCTTCTCT 801 CCTCCCCTAC ACCAACAAAG AGGAATGGCT GCAAGAGCCC 841 AGATCACCCA TTCCGGGTTC ACTCCCCGCC TCCCCAAGTC 881 AGCAGTCCTA GCCCCAAACC AGCCCAGAGC AGGGTCTCTC 921 TAAAGGGGAC TTGAGGGCCT GAGCAGGAAA GACTGGCCCT 961 CTAGCTTCTA CCCTTTGTCC CTGTAGCCTA TACAGTTTAG1001 AATATTTATT TGTTAATTTT ATTAAAATGC TTTAAAAAAA1041 TAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAA

Another human CRABPII polypeptide sequences is available in the NCBIdatabase as accession number CAI16339.1 (GI:55960771). This sequence isprovided below for easy reference as SEQ ID NO:35.

  1 MPNFSGNWKI IRSENFEELL KVLGVNVMLR KIAVAAASKP 41 AVEIKQEGDT FYIKTSTTVR TTEINFKVGE EFEEQTVDGR 81 PCKSLVKWES ENKMVCEQKL LKGEGPKTSW TRELTNDGEL 121 ILTMTADDVV CTRVYVRE

A nucleic acid sequence for this human cellular retinoic acid-bindingprotein 2 (CRABPII) polypeptide is available in the NCBI database asaccession number NM_(—)001199723.1 (GI:315013541). This sequence isprovided below for easy reference as SEQ ID NO:36.

   1 GATTCAAGTG CTGGCTTTGC GTCCGCTTCC CCATCCACTT  41 ACTAGCGCAG GAGAAGGCTA TCTCGGTCCC CAGAGAAGCC  81 TGGACCCACA CGCGGGCTAG ATCCAGAGAA CCTGACGACC 121 CGGCGACGGC GACGTCTCTT TTGACTAAAA GACAGTGTCC 161 AGTGCTCCAG CCTAGGAGTC TACGGGGACC GCCTCCCGCG 201 CCGCCACCAT GCCCAACTTC TCTGGCAACT GGAAAATCAT 241 CCGATCGGAA AACTTCGAGG AATTGCTCAA AGTGCTGGGG 281 GTGAATGTGA TGCTGAGGAA GATTGCTGTG GCTGCAGCGT 321 CCAAGCCAGC AGTGGAGATC AAACAGGAGG GAGACACTTT 361 CTACATCAAA ACCTCCACCA CCGTGCGCAC CACAGAGATT 401 AACTTCAAGG TTGGGGAGGA GTTTGAGGAG CAGACTGTGG 441 ATGGGAGGCC CTGTAAGAGC CTGGTGAAAT GGGAGAGTGA 481 GAATAAAATG GTCTGTGAGC AGAAGCTCCT GAAGGGAGAG 521 GGCCCCAAGA CCTCGTGGAC CAGAGAACTG ACCAACGATG 561 GGGAACTGAT CCTGACCATG ACGGCGGATG ACGTTGTGTG 601 CACCAGGGTC TACGTCCGAG AGTGAGTGGC CACAGGTAGA 641 ACCGCGGCCG AAGCCCACCA CTGGCCATGC TCACCGCCCT 681 GCTTCACTGC CCCCTCCGTC CCACCCCCTC CTTCTAGGAT 721 AGCGCTCCCC TTACCCCAGT CACTTCTGGG GGTCACTGGG 761 ATGCCTCTTG CAGGGTCTTG CTTTCTTTGA CCTCTTCTCT 801 CCTCCCCTAC ACCAACAAAG AGGAATGGCT GCAAGAGCCC 841 AGATCACCCA TTCCGGGTTC ACTCCCCGCC TCCCCAAGTC 881 AGCAGTCCTA GCCCCAAACC AGCCCAGAGC AGGGTCTCTC 921 TAAAGGGGAC TTGAGGGCCT GAGCAGGAAA GACTGGCCCT 961 CTAGCTTCTA CCCTTTGTCC CTGTAGCCTA TACAGTTTAG1001 AATATTTATT TGTTAATTTT ATTAAAATGC TTTAAAAAAA1041 TAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAA

As is known to the skilled artisan, sequence variation can be present inhuman polypeptides, including CRABPII polypeptides. Thus, isoforms ofCRABPII exist. For example, CRABPII isoform CRA has an amino acidsequence that is present in the NCBI database as accession numberEAW52922.1 (GI:119573307). This sequence is provided below for easyreference as SEQ ID NO:37.

  1 MPNFSGNWKI IRSENFEELL KVLGVNVMLR KIAVAAASKP 41 AVEIKQEGDT FYIKTSTTVR TTEINFKVGE EFEEQTVDGR 81 PCKSLVKWES ENKMVCEQKL LKGEGPKTSW TRELTNDGEL 121 ILTMTADDVV CTRVYVRE

A nucleic acid sequence for this CRABPII isoform CRA polypeptide isavailable in the NCBI database as accession number NM_(—)001878.3(GI:315013540). This sequence is provided below for easy reference asSEQ ID NO:38.

   1 GGAGCGGGAG GCGGGGCCAC TTCAATCCTG GGCAGGGGCG  41 GTTCCGTACA GGGTATAAAA GCTGTCCGCG CGGGAGCCCA  81 GGCCAGCTTT GGGGTTGTCC CTGGACTTGT CTTGGTTCCA 121 GAACCTGACG ACCCGGCGAC GGCGACGTCT CTTTTGACTA 161 AAAGACAGTG TCCAGTGCTC CAGCCTAGGA GTCTACGGGG 201 ACCGCCTCCC GCGCCGCCAC CATGCCCAAC TTCTCTGGCA 241 ACTGGAAAAT CATCCGATCG GAAAACTTCG AGGAATTGCT 281 CAAAGTGCTG GGGGTGAATG TGATGCTGAG GAAGATTGCT 321 GTGGCTGCAG CGTCCAAGCC AGCAGTGGAG ATCAAACAGG 361 AGGGAGACAC TTTCTACATC AAAACCTCCA CCACCGTGCG 401 CACCACAGAG ATTAACTTCA AGGTTGGGGA GGAGTTTGAG 441 GAGCAGACTG TGGATGGGAG GCCCTGTAAG AGCCTGGTGA 481 AATGGGAGAG TGAGAATAAA ATGGTCTGTG AGCAGAAGCT 521 CCTGAAGGGA GAGGGCCCCA AGACCTCGTG GACCAGAGAA 561 CTGACCAACG ATGGGGAACT GATCCTGACC ATGACGGCGG 601 ATGACGTTGT GTGCACCAGG GTCTACGTCC GAGAGTGAGT 641 GGCCACAGGT AGAACCGCGG CCGAAGCCCA CCACTGGCCA 681 TGCTCACCGC CCTGCTTCAC TGCCCCCTCC GTCCCACCCC 721 CTCCTTCTAG GATAGCGCTC CCCTTACCCC AGTCACTTCT 761 GGGGGTCACT GGGATGCCTC TTGCAGGGTC TTGCTTTCTT 801 TGACCTCTTC TCTCCTCCCC TACACCAACA AAGAGGAATG 841 GCTGCAAGAG CCCAGATCAC CCATTCCGGG TTCACTCCCC 881 GCCTCCCCAA GTCAGCAGTC CTAGCCCCAA ACCAGCCCAG 921 AGCAGGGTCT CTCTAAAGGG GACTTGAGGG CCTGAGCAGG 961 AAAGACTGGC CCTCTAGCTT CTACCCTTTG TCCCTGTAGC1001 CTATACAGTT TAGAATATTT ATTTGTTAAT TTTATTAAAA1041 TGCTTTAAAA AAATAAAAAA AAAAAAAAAA AAAAAAAAAA 1081 AAAAAAAA

As illustrated herein, CRABPII polypeptides with modified amino acidsequences exhibit different light transmission and emission properties(see the Examples and FIGS. 5-8). Moreover, a number of other CRABPIIpolypeptides can be used as potential pH sensors, which occupy the pKarange from 2.7 to 7.0.

For example, the following modified R111K: C130X: R132X: Y134X:F3X: I9X:S12X: F15X: L19X: V24X: A32X: A35X: A36X: S37X: K38X:P39X: Q45X: T54X:T56X: T57X: V58X: R59X: T61X: E73X: Q74X: V76X:G78X: C81X: M93X: C95X:L121X: M123X CRABPII polypeptide (SEQ ID NO:46) shows amino acidpositions that can readily be modified to achieve desirable lighttransmission and emission properties when complexed with a retinoid orfluorescent dye ligand, as well as desirable stability in response tochanges in temperature and pH.

  1 MPNXSGNWKX IRXENXEELX KVLGXNVMLR KIXVAXXXXX 41 AVEIKXEGDT FYIKXSXXXX TXEINFKVGE EFEXXTXDXR 81 PXKSLVKWES ENKXVXEQKL LKGEGPKTSW TKELTNDGEL 121 IXTXTADDVV XTXVXVREwherein each X is independently a genetically encoded L-amino acid, anaturally-occurring non-genetically encoded L-amino acid, a syntheticL-amino acid or a synthetic D-amino acid.

Similarly, the following modified Q108K; R2X; F16X; Y19X; M20X; I25X;T29X; A33X; Q38X; K40X; I42X; T51X; T53X; S55X; F57X; R58X; Y60X; V62X;F64X; E72X; S76X; L77X; C95X; Q97X; R104X; W106X; L117X; L119X; Q128X;F130X CRBPII polypeptide (SEQ ID NO:47) shows amino acid positions thatcan readily be modified to achieve desirable light transmission andemission properties when complexed with a retinoid or fluorescent dyeligand, as well as desirable stability in response to changes intemperature and pH.

  1 TXDXNGTWEM ESNENXEGXX KALDXDFAXR KIXVRLTXTX 41 VXDQDGDNFK XKXTXTXXNX DXDXTVGVEF DXYTKXXDNR 81 HVKALVTWEG DVLVXVXKGE KENXGXKXWI EGDKLYXEXT 121 CGDQVCRXVX KKKwherein each X is independently a genetically encoded L-amino acid, anaturally-occurring non-genetically encoded L-amino acid, a syntheticL-amino acid or a synthetic D-amino acid.

Thus, the polypeptides described herein can have amino acid sequencescomprised of any available amino acid. Amino acids included in thepeptides can be genetically encoded L-amino acids, naturally occurringnon-genetically encoded L-amino acids, synthetic L-amino acids orD-enantiomers of any of the above. The amino acid notations used hereinfor the twenty genetically encoded L-amino acids and common non-encodedamino acids are conventional and are as shown in Table 3. These aminoacids can be linked together, for example, by peptidyl linkages,intersubunit linkages, or other intersubunit linkages that areconsistent with enzyme-substrate or receptor-ligand bindinginteractions.

TABLE 3 One-Letter Common Amino Acid Symbol Abbreviation Alanine A AlaArginine R Arg Asparagine N Asn Aspartic acid D Asp Cysteine C CysGlutamine Q Gln Glutamic acid E Glu Glycine G Gly Histidine H HisIsoleucine I Ile Leucine L Leu Lysine K Lys Methionine M MetPhenylalanine F Phe Proline P Pro Serine S Ser Threonine T ThrTryptophan W Trp Tyrosine Y Tyr Valine V Val β-Alanine bAla2,3-Diaminopropionic acid Dpr α-Aminoisobutyric acid Aib N-Methylglycine(sarcosine) MeGly Ornithine Orn Citrulline Cit t-Butylalanine t-BuAt-Butylglycine t-BuG N-methylisoleucine MeIle Phenylglycine PhgCyclohexylalanine Cha Norleucine Nle Naphthylalanine Nal Pyridylalanine3-Benzothienyl alanine 4-Chlorophenylalanine Phe(4-Cl)2-Fluorophenylalanine Phe(2-F) 3-Fluorophenylalanine Phe(3-F)4-Fluorophenylalanine Phe(4-F) Penicillamine Pen1,2,3,4-Tetrahydro-isoquinoline- Tic 3-carboxylic acidβ-2-thienylalanine Thi Methionine sulfoxide MSO Homoarginine hArgN-acetyl lysine AcLys 2,4-Diamino butyric acid Dbu ρ-AminophenylalaninePhe(pNH₂) N-methylvaline MeVal Homocysteine hCys Homoserine hSer ε-Aminohexanoic acid Aha δ-Amino valeric acid Ava 2,3-Diaminobutyric acid Dab

Certain amino acids that are not genetically encoded can be present inpolypeptides of the invention including β-alanine (b-Ala) and otheromega-amino acids such as 3-aminopropionic acid (Dap),2,3-diaminopropionic acid (Dpr), 4-aminobutyric acid and so forth;α-aminoisobutyric acid (Aib); ε-aminohexanoic acid (Aha); δ-aminovalericacid (Ava); N-methylglycine (MeGly); ornithine (Orn); citrulline (Cit);t-butylalanine (t-BuA); t-butylglycine (t-BuG); N-methylisoleucine(MeIle); phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle);2-naphthylalanine (2-Nal); 4-chlorophenylalanine (Phe(4-Cl));2-fluorophenylalanine (Phe(2-F)); 3-fluorophenylalanine (Phe(3-F));4-fluorophenylalanine (Phe(4-F)); penicillamine (Pen);1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic);.beta.-2-thienylalanine (Thi); methionine sulfoxide (MSO); homoarginine(hArg); N-acetyl lysine (AcLys); 2,3-diaminobutyric acid (Dab);2,3-diaminobutyric acid (Dbu); p-aminophenylalanine (Phe(pNH₂));N-methyl valine (MeVal); homocysteine (hCys) and homoserine (hSer).

The classifications of the above-described genetically encoded andnon-encoded amino acids are summarized in Table 4, below. It is to beunderstood that Table 4 is for illustrative purposes only and does notpurport to be an exhaustive list of amino acid residues which maycomprise the polypeptides described herein. Other amino acid residueswhich are useful for making the polypeptides described herein can befound, e.g., in Fasman, 1989, CRC Practical Handbook of Biochemistry andMolecular Biology, CRC Press, Inc., and the references cited therein.Amino acids not specifically mentioned herein can be convenientlyclassified on the basis of known behavior and/or their characteristicchemical and/or physical properties as compared with amino acidsspecifically identified.

TABLE 4 Classification Genetically Encoded Genetically Non-EncodedHydrophobic Aromatic F, Y, W Phg, Nal, Thi, Tic, Phe(4- Cl), Phe(2-F),Phe(3-F), Phe(4-F), Pyridyl Ala, Benzothienyl Ala Apolar M, G, PAliphatic A, V, L, I t-BuA, t-BuG, MeIle, Nle, MeVal, Cha, bAla, MeGly,Aib Hydrophilic Acidic D, E Basic H, K, R Dpr, Orn, hArg, Phe(p- Nh₂),DBU, A₂BU Polar Q, N, S, T, Y Cit, AcLys, MSO, hSer Cysteine-Like C Pen,hCys, β-methyl Cys

The colorimetric/fluorescent polypeptides can be complexed with retinaland other dyes either covalently or non-covalently. In some embodiments,the complex between the polypeptide and retinal (or another dye) isnon-covalent. In other embodiments, a covalent bond between thepolypeptide and retinal (or another dye) forms spontaneously by attackof an amino acid in the polypeptide upon an active group in the retinalor dye. For example, when lysine is present at position 108 of thehCRBPII polypeptide (instead of glutamine), such a Q108K hCRBPIIpolypeptide and adopts a favorable three-dimensional structure forpositioning the lysine to attack the retinal aldehyde to form aprotonated Schiff base.

Generating Modified Colorimetric/Fluorescent Polypeptides

The colorimetric/fluorescent polypeptides described herein may besynthesized by methods available in the art, including recombinant DNAmethods and chemical synthesis.

Chemical synthesis may be performed using standard solution phase orsolid phase peptide synthesis techniques, in which a peptide linkageoccurs through the direct condensation of the α-amino group of one aminoacid with the carboxy group of the other amino acid with the eliminationof a water molecule. Peptide bond synthesis by direct condensation, asformulated above, may involve suppression of the reactive character ofthe amino group of the first and of the carboxyl group of the secondamino acid. The masking substituents must permit their ready removal,without inducing breakdown of the labile peptide molecule.

In solution phase synthesis, a wide variety of coupling methods andprotecting groups may be used (see Gross and Meienhofer, eds., “ThePeptides: Analysis, Synthesis, Biology,” Vol. 1-4 (Academic Press,1979); Bodansky and Bodansky, “The Practice of Peptide Synthesis,” 2ded. (Springer Verlag, 1994)). In addition, intermediate purification andlinear scale up are possible. Those of ordinary skill in the art willappreciate that solution synthesis requires consideration of main chainand side chain protecting groups and activation method. In addition,careful segment selection may be necessary to minimize racemizationduring segment condensation. Solubility considerations are also afactor.

Solid phase peptide synthesis uses an insoluble polymer for supportduring organic synthesis. The polymer-supported peptide chain permitsthe use of simple washing and filtration steps instead of laboriouspurifications at intermediate steps. Solid-phase peptide synthesis maygenerally be performed according to the method of Merrifield et al., J.Am. Chem. Soc. 85:2149, 1963, which involves assembling a linear peptidechain on a resin support using protected amino acids. Solid phasepeptide synthesis typically utilizes either the Boc or Fmoc strategy,which are now well known in the art.

Those of ordinary skill in the art will recognize that, in solid phasesynthesis, deprotection and coupling reactions must go to completion andthe side-chain blocking groups must be stable throughout the entiresynthesis. In addition, solid phase synthesis is generally most suitablewhen peptides are to be made on a small scale.

The modified iLBP colorimetric/fluorescent polypeptides described hereinmay be synthesized by recombinant DNA methods. Therefore, another aspectof the invention is a nucleic acid encoding modified iLBPcolorimetric/fluorescent polypeptides described herein.

As used herein, the term “isolated” refers to a nucleic acid,polypeptide or amino acid (or other component) that is removed from atleast one component with which it is naturally associated. The isolatednucleic acid, polypeptide or amino acid (or other component) can, butneed not, be purified. Instead, the isolated nucleic acid, polypeptideor amino acid (or other component), while not within its naturalenvironment, may be present in another environment, for example, a hostcell that normally does not have such an isolated nucleic acid,polypeptide or amino acid (or other component).

Modifications to the amino acid sequences of thecolorimetric/fluorescent polypeptides can be preparing a modifiednucleic acid that encodes the colorimetric/fluorescent polypeptide. Theterm “modified nucleic acid” herein refers to a DNA or RNA that has beenaltered to contain at least one mutation to encode a modified iLBPcolorimetric/fluorescent polypeptide.

Several methods are known in the art that are suitable for generatingmodified nucleic acids, including but not limited to site-saturationmutagenesis, scanning mutagenesis, insertional mutagenesis, deletionmutagenesis, random mutagenesis, site-directed mutagenesis, anddirected-evolution, as well as various other recombinatorial approaches.The commonly used methods include DNA shuffling (Stemmer W P, Proc NatlAcad Sci USA. 25; 91(22):10747-51 [1994]), methods based onnon-homologous recombination of genes e.g. ITCHY (Ostermeier et al.,Bioorg Med. Chem. 7(10):2139-44 [1999]), SCRACHY (Lutz et al. Proc NatlAcad Sci USA. 98(20):11248-53 [2001]), SHIPREC (Sieber et al., Nat.Biotechnol. 19(5):456-60 [2001]), and NRR (Bittker et al., Nat.Biotechnol. 20(10):1024-9 [2001]; Bittker et al., Proc Natl Acad SciUSA. 101(18):7011-6 [2004]), and methods that rely on the use ofoligonucleotides to insert random and targeted mutations, deletionsand/or insertions (Ness et al., Nat. Biotechnol. 20(12):1251-5 [2002];Coco et al., Nat. Biotechnol. 20(12):1246-50 [2002]; Zha et al.,Chembiochem. 3; 4(1):34-9 [2003], Glaser et al., J. Immunol.149(12):3903-13 [1992], Sondek and Shortie, Proc Natl Acad Sci USA89(8):3581-5 [1992], Yanez et al., Nucleic Acids Res. 32(20):e158[2004], Osuna et al., Nucleic Acids Res. 32(17):e136 [2004], Gaytan etal., Nucleic Acids Res. 29(3):E9 [2001], and Gaytan et al., NucleicAcids Res. 30(16):e84 [2002]).

In some embodiments, the modified nucleic acid encodes an amino acidsubstitution at least at one amino acid position selected from positions1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 103, 104, 105, 106, 107, 108, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,125, 126, 127, 128, 129, 130, 131, 132, 133, 134, of an ILBPpolypeptide, for example, a polypeptide with any of SEQ ID NO:1, 3, 5,6-29, 31, 33, 35, 37, 39-46 and 47. In some embodiments, the modifiedcolorimetric/fluorescent polypeptides is at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 97%, at leastabout 98%, at least about 99% identical to any of the iLBP polypeptidesreferred to herein, including those with SEQ ID NO:1, 3, 5, 6-29, 31,33, 35, 37, 39-44 and 45.

As is known by one with skill in the art, the genetic code is“degenerate,” meaning that several trinucleotide codons can encode thesame amino acid. This degeneracy is apparent from Table 5.

TABLE 5 Second Position 1^(st) 3^(rd) Position T C A G Position T TTT =Phe TCT = Ser TAT = Tyr TGT = Cys T T TTC = Phe TCC = Ser TAC = Tyr TGC= Cys C T TTA = Leu TCA = Ser TAA = Stop TGA = Stop A T TTG = Leu TCG =Ser TAG = Stop TGG = Trp G C CTT = Leu CCT = Pro CAT = His CGT = Arg T CCTC = Leu CCC = Pro CAC = His CGC = Arg C C CTA = Leu CCA = Pro CAA =Gln CGA = Arg A C CTG = Leu CCG = Pro CAG = Gln CGG = Arg G A ATT = IleACT = Thr AAT = Asn AGT = Ser T A ATC = Ile ACC = Thr AAC = Asn AGC =Ser C A ATA = Ile ACA = Thr AAA = Lys AGA = Arg A A ATG = Met ACG = ThrAAG = Lys AGG = Arg G G GTT = Val GCT = Ala GAT = Asp GGT = Gly T G GTC= Val GCC = Ala GAC = Asp GGC = Gly C G GTA = Val GCA = Ala GAA = GlnGGA = Gly A G GTG = Val GCG = Ala GAG = Gln GGG = Gly G

Hence, many changes in the nucleotide sequence of the isolated nucleicacids described herein may be silent and may not alter the amino acidsequence encoded by the nucleic acid. Where nucleic acid sequencealterations are silent, an isolated nucleic acid will encode apolypeptide with the same amino acid sequence as the reference nucleicacid. Therefore, a particular nucleic acid sequence of the inventionalso encompasses variants with degenerate codon substitutions, andcomplementary sequences thereof, as well as the sequence explicitlyspecified by a SEQ ID NO. Specifically, degenerate codon substitutionsmay be achieved by generating sequences in which the reference codon isreplaced by any of the codons for the amino acid specified by thereference codon. In general, the third position of one or more selectedcodons can be substituted with mixed-base and/or deoxyinosine residuesas disclosed by Batzer et al., Nucleic Acid Res., 19, 5081 (1991) and/orOhtsuka et al., J. Biol. Chem., 260, 2605 (1985); Rossolini et al., Mol.Cell. Probes, 8, 91 (1994).

The modified nucleic acid can be operably linked to one or more nucleicacid segments that encode one or more regulatory elements. Such aconstruct is referred to as an “expression cassette.”

The term “regulatory element,” as used herein, refers to any nucleicacid segment with a sequence that influences transcription ortranslation initiation and rate, or stability and/or mobility of atranscript or polypeptide product. Regulatory element sequences include,but are not limited to, promoters, promoter control elements, proteinbinding sequences, 5′ and 3′ UTRs, transcriptional start sites,termination sequences, polyadenylation sequences, introns, certainsequences within amino acid coding sequences such as secretory signals,protease cleavage sites, and combinations thereof.

The expression cassettes comprising a modified nucleic acid that encodesa modified iLBP polypeptide can be included within a vector tofacilitate manipulation, maintenance, replication, and/or expansion ofthe modified nucleic acids as well as expression polypeptides encodedwithin the modified iLBP polypeptides.

The vector backbone can be any of those employed in the art such asplasmids, viruses, artificial chromosomes, BACs, YACs and PACs andvectors of the sort described by (a) BAC: Shizuya et al., Proc. Natl.Acad. Sci. USA 89: 8794-8797 (1992); Hamilton et al., Proc. Natl. Acad.Sci. USA 93: 9975-9979 (1996); [0183] (b) YAC: Burke et al., Science236:806-812 (1987); (c) PAC: Sternberg N. et al., Proc Natl Acad SciUSA. January; 87(1):103-7 (1990); (d) Bacteria-Yeast Shuttle Vectors:Bradshaw et al., Nucl Acids Res 23: 4850-4856 (1995); (e) Lambda PhageVectors: Replacement Vector, e.g., Frischauf et al., J. Mol. Biol 170:827-842 (1983); or Insertion vector, e.g., Huynh et al., In: Glover N M(ed) DNA Cloning: A practical Approach, Vol. 1 Oxford: IRL Press (1985);T-DNA gene fusion vectors: Walden et al., Mol Cell Biol 1: 175-194(1990); and (g) Plasmid vectors: Sambrook et al., infra.

Retinoid Ligands

The modified iLBPs of the invention bind retinoid ligands yielding aiLBP-retinoid complexes that absorb and transmit light. Retinoids are aclass of chemical compounds that are related chemically to vitamin A.Such retinoids include retinal, retinol, tretinoin, isotretinoin,etretinate, acitretin, carotenoid, vitamin A, and retinoic acid.

Vitamin A is metabolized into the light-absorbing molecule retinal,which is needed by animals for both low-light (scotopic vision) andcolor vision. The major form of vitamin A in food from animal sources isan ester, for example, retinyl palmitate. The vitamin A ester isconverted to retinol in the small intestine, which functions as astorage form of the vitamin, and which can be converted to and from itsvisually active aldehyde form, retinal. Retinoic acid is a metabolitethat can be irreversibly synthesized from vitamin A, but it has onlypartial vitamin A activity.

Retinoids have a ring to which a isoprenoid chain, called a retinylgroup, is attached. In some embodiments, the ring is an aromatic ring.In other embodiments, the ring is a beta-ionone ring to which anisoprenoid chain is attached. Both the ring and the isoprenoid chain areneeded for vitamin activity. The orange pigment ofcarrots—beta-carotene—can be represented as two connected retinylgroups, which are used in the body to contribute to vitamin A levels.Alpha-carotene and gamma-carotene also have a single retinyl group,which give them some vitamin activity. None of the other carotenes havevitamin activity. The carotenoid beta-cryptoxanthin possesses an iononegroup and has vitamin activity in humans. The structures of retinoicacid and retinol are shown below.

Retinal is also called retinaldehyde or vitamin A aldehyde. It is apolyene chromophore that binds to proteins called opsins. The structureof all-trans-retinal is shown below.

All of the retinoids and related vitamin A-like molecules can be used asligands for the modified iLBPs of the invention of the invention inorder to form fluorescent and colorimetric labeling agents. Thus, theretinoid molecule is added or administered to the modified iLBPs of theinvention of the invention, which bind the retinoid ligands, and therebyform fluorescent and colorimetric labeling agents. In general themodified iLBPs of the invention of the invention bind retinal as theretinoid ligand molecule in order to absorb and transmit light. However,in some embodiments, retinol, tretinoin, isotretinoin, etretinate,acitretin, carotenoid, vitamin A, retinoic acid or other vitamin A-likemolecules are used, added or administered either because the modifiedpolypeptide can bind those retinoids or because such retinoids can beconverted into another retinoid (e.g., retinal) by cellular enzymes.

Other Dye Ligands

The invention also relates to dye ligand compounds that can bind themodified iLBP polypeptides described herein, for example, via formationof a Schiff base formed between an amino group in the iLBP polypeptideand an aldehyde (—CHO) on the dye ligand molecule. The iLBP-dye ligandcomplex transmits light and/or is fluorescent.

Thus, one aspect of the invention is a dye ligand of formula I:

Ring-Y—CHO

wherein:

-   -   Ring is an optionally substituted C₅-C₁₄ mono-, di- or tricyclic        cycloalkyl, aryl or heterocyclic ring, wherein the heterocyclic        ring has at least one nitrogen or oxygen ring atom, and wherein        the Ring has 1-3 optional substituents that are selected from        the group consisting of alkyl, halogen, alkoxy, amino and        sulfhydryl; and    -   Y is a divalent C₂-C₁₂ alkenylene chain that optionally        substituted with 1-3 alkyl groups.

In other embodiments, the merocyanine dye is a compound of formula II:

Ar₁—Y₁—CHO

wherein:

-   -   Ar₁ is a C₅-C₁₀ mono- or dicyclic heterocyclic ring system, with        at least one nitrogen or oxygen ring atom; and    -   Y₁ is a divalent C₂-C₁₂ alkenylene chain that is optionally        substituted with 1-3 alkyl groups.

In some embodiments, Ring is monocyclic. In other embodiments, the Ringis bicyclic. When the Ring is a heterocyclic ring it can be a mono-, di-or tricyclic heteroaryl ring, where at least one of the rings in theheteroaryl ring is aromatic.

The alkenylene chain can include a —(CH═CH)_(n)— chain where n is aninteger of from 1 to 6, and where the alkenylene chain can besubstituted with 1-3 alkyl groups, where the alkyl groups have from 1 toabout 10 carbon atoms, and typically from 1 to 6 carbons or, in someembodiments, from 1 to 3 carbon atoms.

One example of a merocyanine dye that can be used with thecolorimetric/fluorescent proteins described herein has the followingstructure.

When the dye binds to a modified iLBP polypeptide it can form a Schiffbase that is pH sensitive as shown below.

As illustrated, this compound can form a Schiff base with the protein,where the Schiff base is protonated at acidic pH and not protonated atbasic pH.

Fusion Proteins

The colorimetric/fluorescent polypeptides described herein can be fusedto any molecule or fusion partner of interest. The combination of thecolorimetric/fluorescent polypeptide and the fusion partner is referredto as a “fusion protein” even if a portion of the fusion protein is nota polypeptide.

The terms “fusion protein” and “chimeric protein,” as used herein, areinterchangeable and refer to polypeptides and proteins which comprise acolorimetric/fluorescent polypeptides described herein and a fusionpartner. In some embodiments, a linker can join thecolorimetric/fluorescent polypeptide and the fusion partner. In otherembodiments, the colorimetric/fluorescent polypeptide and the fusionpartner fused directly together. When the fusion partner is a protein,it may be fused in frame, for example, to facilitate recombinantsynthesis or allow the fusion protein to be made in vivo.

Fusion partners can include any naturally occurring, or synthetic,molecule, component or material. Examples of fusion partners ormolecules to which the colorimetric/fluorescent polypeptides describedherein can be fused include biological molecules, small syntheticmolecules, proteins, antibodies, antibody fragments, nucleic acids,polysaccharides, glycans, therapeutic agents, drugs, pharmaceuticals,ligands, cofactors, vitamins, polymers, intracellular molecules,extracellular molecules, viruses, viral components, subcellularstructures, cellular organelles, cells, neurons, axons, dendrites,membranes, secreted factors, secreted materials, toxins, waste products,dyes, labels and the like.

In a further embodiment, a fusion protein may comprise more than onecolorimetric/fluorescent polypeptides and/or more than one fusionpartner. In these embodiments, the multiple colorimetric/fluorescentpolypeptides may be the same or different, the multiple fusion partnersmay be the same or different. One or more linkers can be used to jointhe colorimetric/fluorescent polypeptides and/or fusion partners.

In one embodiment, fusion partner is biologically active. Examples offusion partners include, but are not limited to, interleukin (IL)-11,thymosin β4, thymosin α1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-10, IL-13, IL-15, IL-18, Protease-activated receptor 1 (PAR1), PAR3,PAR4, RANTES, stromal cell-derived factor-1α, monocyte chemotacticprotein, stem cell factor, FLT-3L, parathyroid hormone, thrombopoietin,epidermal growth factor, basic fibroblast growth factor, insulin-likegrowth factor, granulocyte-macrophage colony stimulating factor,granulocyte colony stimulating factor, macrophage colony stimulatingfactor, platelet-derived growth factor, transforming growth factor(TGF)-β1, tumor necrosis factor (TNF)-α, interferon (IFN)-α, IFN-γ,hepatocyte growth factor, vascular endothelial growth factor, animmunoglobulin heavy chain, an immunoglobulin light chain and othermolecules of interest to those of skill in the art.

In some embodiments, the fusion partner is a target protein, where thetarget protein is a biological molecule whose in vivo location, functionand/or activity is of interest to one of skill in the art. For example,the target protein can be any fusion partner described herein

Fusion Protein Synthesis

The colorimetric/fluorescent polypeptide and the fusion partner can besynthetically, recombinantly, or chemically fused.

In some embodiments, the modified iLBP colorimetric/fluorescentpolypeptide and the fusion partner are recombinantly fused by joining orligating a nucleic acid that encodes the modified iLBP polypeptide witha nucleic acid that encodes the fusion partner.

The nucleic acids coding for the colorimetric/fluorescent polypeptideand the fusion partner are isolated, synthesized or otherwise obtainedand fused in frame together to form a hybrid nucleic acid containing thecoding region for the colorimetric/fluorescent polypeptide and thecoding region for the fusion partner. In one embodiment, the nucleicacids are ligated together using a ligase. The hybrid nucleic acid canthen be operably linked to nucleic acids encoding regulatory elements.The term “operably linked,” as used herein, refers to a regulatoryelement being linked to a nucleic acid encoding acolorimetric/fluorescent polypeptide, fusion partner or fusion proteinin such a manner that the regulatory element exerts an effect on thetranscription and/or translation of the nucleic acid.

The nucleic acid(s) encoding a colorimetric/fluorescent polypeptide,fusion partner or fusion protein can be placed, maintained, replicated,or amplified within a vector (e.g., a plasmid, virus, or bacteriophagevector). In one embodiment, the vector is an expression vector. Thevector can be autonomously replicable in a host cell. The vector canalso contain a selectable marker. Selectable markers include nucleicacids encoding drug resistance (e.g., ampicillin or tetracycline), anenzyme activity, an auxotrophy complement or an inert protein that maybe detected in a host cell by methods known in the art. For example, theselectable marker may be green fluorescent protein that may be detectedupon expression in a host cell by visualization through light microscopyunder ultra-violet light.

The vector can include nucleic acid segments encoding regulatorysequences (e.g., transcription and translation elements) to controlexpression of the colorimetric/fluorescent polypeptide, fusion partneror fusion protein in a suitable host cell. The regulatory sequences mayinclude one or more of promoter regions, enhancer regions, transcriptiontermination sites, ribosome binding sites, initiation codons, splicesignals, introns, polyadenylation signals, Shine/Dalgarno translationsequences, and Kozak consensus sequences. Regulatory sequences arechosen with regard to the host cell in which thecolorimetric/fluorescent polypeptide, fusion partner or fusion proteinis to be produced. Suitable bacterial promoters include, but are notlimited to, bacteriophage λpL or pR, T6, T7, T7/lacO, lac, recA, gal,trp, ara, hut, and trp-lac. Suitable eukaryotic promoters include, butare not limited to, PRBI, GAPDH, metallothionein, thymidine kinase,viral LTR, cytomegalovirus, SV40, or tissue-specific or tumor-specificpromoters such as α-fetoprotein, amylase, cathepsin E, M1 muscarinicreceptor, or γ-glutamyl transferase.

Colorimetric/fluorescent polypeptides, fusion partners or fusionproteins that are designed to be secreted from a host cell into theculture medium or into the periplasm of the host cell may also contain asignal sequence. The signal sequence may be the fusion partner or may bein addition to the fusion partner. A nucleic acid encoding a signalsequence may be operably linked to the 5′ end of the nucleic acidencoding the colorimetric/fluorescent polypeptide, fusion partner orfusion protein. Suitable signal sequences are available in the art andinclude, for example, MBP, GST, TRX, DsbA, and LamB from E. coli andα-factor from yeast.

In some embodiments, the vector can also comprise one or more cloningsites, e.g., restriction enzyme recognition sites, upstream and/ordownstream of the nucleic acid(s) encoding a colorimetric/fluorescentpolypeptide, fusion partner or fusion protein to facilitate the cloningof these nucleic acid(s). Examples of suitable expression vectors arefound in U.S. Pat. No. 5,814,503, which is incorporated herein byreference.

Another aspect of the invention is a method of preparing a nucleic acidencoding a colorimetric/fluorescent polypeptide, fusion partner orfusion protein, comprising inserting a nucleic acid encoding fusionpartner into a cloning site of a vector such that the fusion partnernucleic acid is upstream or downstream and in frame with a nucleic acidencoding a colorimetric/fluorescent polypeptide.

Another aspect of the invention is a host cell comprising a vectorencoding a colorimetric/fluorescent polypeptide, a fusion partner or afusion protein. The host cell may be any cell suitable for expression ofa colorimetric/fluorescent polypeptide, a fusion partner or fusionprotein, including prokaryotic (e.g., bacterial) and eukaryotic (e.g.,fungi, yeast, animal, insect, plant) cells. Suitable prokaryotic hostcells include, but are not limited to, E. coli (e.g., strains DHS,HB101, JM109, or W3110), Bacillus, Streptomyces, Salmonella, Serratia,and Pseudomonas species. Suitable eukaryotic host cells include culturedeukaryotic cells as well as cells administered to a eukaryotic organism.Examples include cultured mammalian cells, cancer cells, non-cancerouscells, healthy primary cultured cells, cells isolated from mammaliantissues, yeast, COS, CHO, HepG-2, CV-1, LLC-MK₂, 3T3, HeLa, RPMI8226,293, BHK-21, Sf9, Saccharomyces, Pichia, Hansenula, Kluyveromyces,Aspergillus, or Trichoderma species.

Methods and materials for preparing recombinant vectors and transforminghost cells using the same, replicating the vectors in host cells andexpressing biologically active foreign polypeptides and proteins aredescribed in Old et al., Principles of Gene Manipulation, 2nd edition,(1981); Sambrook et al., Molecular Cloning, 3rd edition, Cold SpringHarbor Laboratory, 2001, and Ausubel et al., Current Protocols inMolecular Biology, John Wiley & Sons, New York 3rd edition, (2000), eachincorporated herein by reference.

Vectors may be introduced into a host cell by any means known in theart, including, but not limited to, transformation, calcium phosphateprecipitation, electroporation, lipofection, microinjection, and viralinfection.

Another aspect of the invention is a method that involves propagatingthe modified nucleic acid in a prokaryotic or eukaryotic cell.

Such a method can also or separately involve producing acolorimetric/fluorescent modified iLBP polypeptide, a fusion partner orfusion protein. Such a method can include preparing a vector comprisinga nucleic acid encoding a colorimetric/fluorescent polypeptide, a fusionpartner and/or a fusion protein, delivering the vector into a host cell,culturing the host cell under conditions in which the acolorimetric/fluorescent polypeptide, a fusion partner and/or fusionprotein is expressed, and isolating the colorimetric/fluorescentpolypeptide, fusion partner and/or fusion protein.

The colorimetric/fluorescent modified iLBP polypeptide, fusion partneror fusion protein may be separated from the host cell by any means knownin the art. If the colorimetric/fluorescent polypeptide, fusion partneror fusion protein is secreted from the host cell, the culture mediumcontaining the colorimetric/fluorescent polypeptide, fusion partner orfusion protein may be collected. If the colorimetric/fluorescentpolypeptide, fusion partner or fusion protein is not secreted from thehost cell, the cell may be lysed to release the colorimetric/fluorescentpolypeptide, fusion partner or fusion protein. For example, bacterialcells may be lysed by application of high pressure (e.g., with a highpressure homogenizer) or by sonication.

Method of Observing Target Molecules In Vivo

According to the invention, target molecules can be observed in vivo bya variety of methods. For example, the target molecule can be observedby detecting the light transmitted or emitted by a modified iLBPprotein:retinoid/dye complex when the modified iLBP protein:retinoid/dyecomplex is associated with, or fused to, a selected target molecule.Thus, in some embodiments a living cell that includes a modified nucleicacid is generated where the modified nucleic acid encodes a fusionprotein that includes a fusion protein comprising the modified iLBPpolypeptide of the invention fused in frame with the target protein.Upon expression of the fusion protein, and addition of a retinoid or dyeligand to the cell, a colorimetric or fluorescent signal is readilydetected so that the location, function and/or activity of the targetprotein can be observed.

Therefore, another aspect of the invention is a method of observing atarget protein in vivo comprising contacting a living cell with aretinoid or dye that binds a modified polypeptide encoded by theisolated nucleic acid of claim 1, wherein the cell expresses a fusionprotein comprising the modified polypeptide fused in frame with thetarget protein.

Thus, a modified nucleic acid can be expressed in an animal cell byinserting the modified nucleic acid into the animal cell (e.g., into thegenome of the cell), where the modified nucleic acid encodes a fusionprotein comprising the modified polypeptide fused in frame with thetarget protein, and where the modified nucleic acid is operably linkedto a nucleic acid segment encoding at least one regulatory element thatpromotes expression of the fusion protein. After construction of theanimal cell containing such a modified nucleic acid, the cell can becultured or replicated as desired. When initiating a study involvingobserving the location, function and/or activity of the target proteinwithin the cell, the cell is exposed to, or contacted with, a retinoidor fluorescent dye that can bind to the modified iLBP polypeptide fusedto the target protein.

Another embodiment includes a method for providing an expressioncassette or vector that encodes one of the modified iLBP polypeptidedescribed herein, comprising, offering a retinoid or dye plus theexpression cassette or vector for sale to a customer along with theright to use retinoid or dye and the expression cassette or vector.

Kits

Another embodiment of the invention is a kit that includes at least onecontainer comprising a nucleic acid encoding a modified iLBPpolypeptide, where the modified polypeptide transmits or emits lightwhen bound to a retinoid or fluorescent dye molecule, and where theintracellular lipid binding protein has been modified so that an aminoacid at any of positions 102-135 can form a Schiff base with an aldehydeon a retinoid or dye ligand. In some embodiments, the nucleic acidencoding the modified iLBP polypeptide is operably linked to at leastone nucleic acid encoding regulatory element. In other embodiments, anexpression cassette including the nucleic acid encoding the modifiediLBP polypeptide is present within the container of the kit. In furtherembodiments, a vector comprising the expression cassette that includesthe nucleic acid encoding the modified iLBP polypeptide is presentwithin the container of the kit.

The nucleic acid encoding the modified iLBP polypeptide can also includerestriction enzyme cleavage sites to facilitate fusion of nucleic acidsencoding other peptides and polypeptides (e.g., a fusion partner).Preferably, the restriction enzyme cleavage sites are positioned so thata selected nucleic acid can be joined in-frame to the cleavage site. Thekit can therefore also include a container comprising a restrictionenzyme for cleaving the nucleic acid encoding the modified iLBPpolypeptide. In addition, the kit can include a container comprising anenzyme for joining or ligating the nucleic acid encoding the modifiediLBP polypeptide with a selected nucleic acid (e.g., a nucleic acidencoding a fusion partner).

Instructions for manipulating and/or using the nucleic acid encoding themodified iLBP polypeptide can also be provided in the kit.

Other containers and materials can be present within the kit. Forexample, the kit can include at least one container comprising aretinoid or a dye ligand. The kit can contain primers for amplifying orfurther modifying the sequence of the nucleic acid encoding the modifiediLBP polypeptide. The kit can include a container comprising Dpn Iendonuclease for specifically cleaving methylated and/or hemimethylatedDNA. The kit can include a container with host cells that can betransformed with the nucleic acid encoding the modified iLBPpolypeptide, or an expression cassette or vector comprising nucleic acidencoding the modified iLBP polypeptide. The kit can also includematerials for purifying or precipitating nucleic acids (e.g., aftercleavage, ligation or other manipulations) and/or materials forpurifying and/or concentrating a modified iLBP polypeptide or a fusionprotein comprising a modified iLBP polypeptide. The kit can also includesolutions for dissolving or suspending a modified iLBP polypeptideand/or a fusion protein.

Another aspect of the invention is a kit that includes at least one acontainer comprising a modified iLBP polypeptide. This kit can alsoinclude at least one container comprising a retinoid or a dye ligand. Insome embodiments, the kit can include reagents for fusing the modifiediLBP polypeptide to another molecule of interest (e.g., a fusionpartner). The kit can also include materials or solutions for purifying,dissolving or suspending the modified iLBP polypeptide and/or a fusionprotein.

Instructions for manipulating and/or using the modified iLBP polypeptidecan also be provided in the kit.

The following non-limiting examples illustrate certain aspects of theinvention and some of the methods used in the development of theinvention.

Example 1 Materials and Methods

This Example describes some of the materials and methods used indeveloping the invention.

Generation of CRBP Mutant Polypeptides

CRBP mutants were made using “Quick Change” mutagenesis procedures,although no commercial kit was employed. A double-stranded DNA vectorencoding a selected CRBP sequences was prepared, as well as twosynthetic oligonucleotide primers containing the desired mutation. Theends of the oligonucleotide primers also contained DNA that wascomplementary to opposite strands of the vector. The CRBP-containing DNAvector was extended using the mutant primers and a thermally stable DNApolymerase (e.g., PfuTurbo® DNA polymerase) during polymerase chainreaction (PCR) thermal cycling. By incorporation of the oligonucleotideprimers into the CRBP-containing DNA vector, a mutated plasmidcontaining staggered nicks was generated. After this primer-extensionreaction, the product is treated with Dpn I endonuclease (targetsequence: 5′-Gm6ATC-3′), which specifically cleaves methylated andhemimethylated DNA. DNA isolated from E. coli is dam-methylated and issusceptible to Dpn I digestion. Cleavage with Dpn I endonucleasetherefore digested the parental DNA template that is methylated but notthe mutated CRBP DNA that was synthesized by primer-extension.

The nicked DNA plasmids containing the desired CRBP mutations were thentransformed into E. coli host cells for expression of the mutant CRBPprotein.

Binding of Retinal and Merocyanine Dyes to the CRBP Polypeptides

The modified CRBP polypeptides were mixed with retinal, typically at astoichiometric ratio of about 2:1 and the absorption/transmission oflight by these retinal:CRBP complexes was measured using a Cary 300 BioWinUV, Varian spectrometer.

In addition, a merocyanine dye with the following structure was mixedwith the modified CRBP polypeptides, typically at a molar ratio of about1:2.

The light absorption and transmission properties of the CRBP:merocyaninedye were also measured by obtaining ultraviolet-visible range spectra ofthese complexes using a Cary 300 Bio WinUV, Varian spectrometer or, forfluorescence, a Fluorolog-3 spectrometer.

Example 2 The Modified CRBP Polypeptides Transmit Light at a Variety ofWavelengths

This Example illustrates that modified CRBP polypeptides transmit lightat a variety of wavelengths when combined with a retinoid or fluorescentdye ligand.

A variety of CRBP mutants were expressed in separate aliquots of E. colihost cells, and the mutant CRBP proteins were isolated by ion exchangechromatography. The CRBP protein preparations were suspended inphosphate buffered saline (PBS). Retinal at a molar ratio of about 1:2was mixed with each mutant CRBP preparation and an ultraviolet-visiblerange spectrum of each CRBP:retinal complex was obtained.

As shown in FIG. 1A, mutations in the human CRBP gene give rise topolypeptide products that transmit light at different wavelengths whenthe mutant CRBP polypeptide is complexed with retinal (other dyes canalso be used). For example, a modified human CRBPII polypeptide withlysine at position 108 instead of glutamine (i.e., a Q108K hCRBPIIpolypeptide) maximally absorbs light at 506 nm. Other human CRBPIIpolypeptides with various amino acid substitutions exhibit modifyinglight absorption and transmission properties. Thus, the followingmodified hCRBPII polypeptides have the indicted maximum wavelengths ofabsorption: Q108K:T51D (λmax=474 nm); Q108K:K40L:Y60W (λmax=512 nm);Q108K:K40L:R58F (λmax=524 nm); Q108K:K40L:R58Y (λmax=535 nm);Q108K:K40L:R58Y,T51V (λmax=563); Q108K:K40L:R58W:T51V:T53C (λmax=585nm); 108K:K40L:R58W:T51V: T53C:T291L:Y19W (λmax=591 nm);Q108K:K40L:R58W:T51V:T53C:T29L: Y19W:Q4W (λmax=613 nm);Q108K:K40L:R58W:T51V:T53C:T29L:Y19W:Q4R (λmax=622 nm);Q108K:K40L:R58W:T51V:T53C:T29L:Y19W:Q4R:A33W (λmax=644 nm).

Examination of an x-ray crystal structure of the hCRBPII polypeptideshows that this Q108K hCRBPII modified polypeptide adopts a favorablethree-dimensional structure for positioning the lysine to attack theretinal aldehyde to form a protonated Schiff base. This Schiff base canbe stabilized or de-stabilized by other amino acids that are naturallypresent in the hCRBPII polypeptide structure, or that replace thenatural amino acids. Thus, the folding of the Q108K hCRBPII modifiedpolypeptide brings a lysine residue at about position 40 close to theSchiff base that forms between the retinal aldehyde and the nitrogen ofthe lysine at position 108. This lysine at position 40 perturbs the pKaof the protonated Schiff base. However, the pKa can be restored byintroduction of a counter ion or replacement of the lysine. For example,when the lysine at position 40 is replaced with a leucine, a modifiedQ108K; K40L hCRBPII polypeptide is formed, which in combination withretinal maximally absorbs light is 508 nm.

Example 3 Modified CRBP Polypeptides Transmit Light and/or Fluoresce InVivo

This Example illustrates that the signal provided by modified CRBPpolypeptides is not masked by other polypeptides or factors in livingcells and is sufficiently strong and distinct to be useful for in vivostudies.

A variety of CRBP mutants were expressed in E. coli cells. Retinal wasthen added to the separate cell preparations containing different CRBPmutant polypeptides to ascertain whether the cells expressing thesepolypeptides would strongly exhibit distinctive colors. As demonstratedby FIG. 2A, the proteins clearly and specifically color the cells,indicating that these proteins provide sufficient signal to be usefulfor detecting the expression of proteins in living cells. Moreover, thedifferent modifications in the CRBP polypeptides give rise to lighttransmission in a variety of distinct colors. Thus, different modifiedCRBP proteins can be employed at the same time to observe differentbiological functions when the different CRBP polypeptides are expressedin vivo as fusion proteins joined to selected biological products.

FIG. 2B shows that CRBP mutant polypeptides are clearly seen when boundto a standard chromatography column and that each of the colors areclearly distinct and readily identifiable. This shows that proteinsfused with the mutant CRBP polypeptides can be seen at a glance duringcolumn chromatography and that many different proteins could besimultaneously observed. For example it would be possible to identifyprotein complexes and sub-complexes visually as they are separatedchromatographically.

In another experiment, the following pH sensitive merocyanine dye wasused for detecting fluorescence in living cells.

A selected cellular retinol binding protein (CRBP) mutant polypeptidewas expressed in E. coli, the merocyanine fluorescent dye shown abovewas added and a robust fluorescence signal was observed in bacterialcells without significant background. Cells with only the merocyaninedye exhibit essentially no fluorescence (left frame, FIG. 3A). Cellsthat express the wild type CRBP protein, which does not bind themerocyanine dye also exhibit no fluorescence (center frame, FIG. 3B).However, a robust fluorescence signal is clearly visible when cellsexpressing a modified CRBP protein that binds the dye are mixed with themerocyanine dye (right frame, FIG. 3C).

These data indicate that the merocyanine dye can penetrate bacterialcells and interact with the CRBP polypeptide as a ligand that binds thepolypeptide in vivo. These data also show that background fluorescencefrom the dye ligand alone does not pose a significant problem.Fluorescence is achieved in this system in a matter of minutes afteraddition of the ligand, indicating that both transport of the dye ligandinto the cell and binding are quite rapid.

Example 4 CRBP Fusion Proteins Fluoresce in Mammalian Cells

This Example describes fusions between CRBP and Green Fluorescentprotein (GFP), some of which are also fused to retinoblastoma protein(RB). The RB protein directs the fusion protein to the nucleus. Asillustrated below and in the figures, the CRBP proteins exhibit strongfluorescence even when fused to other proteins.

A fusion protein was prepared by fusing a nucleic acid encoding the GFPin frame to a nucleic acid encoding a selected Q108K:K40L:T51V:R58F CRBPmutant (see schematic diagram in FIG. 4A). The sequence of thisQ108K:K40L:T51V:R58F CRBP polypeptide is as follows (SEQ ID NO:39).

  1 TRDQNGTWEM ESNENFEGYM KALDIDFATR KIAVRLTQT L  41 VIDQDGDNFK  VKTTSTF F NY DVDFTVGVEF DEYTKSLDNR  81 HVKALVTWEG DVLVCVQKGE KENRGWK KWI EGDKLYLELT 121 CGDQVCRQVF KKK

The sequence of this Q108K; K40L; T51V; R58F hCRBPII polypeptide with amethionine at the N-terminus is as follows (SEQ ID NO:40).

  1 MTRDQNGTWE MESNENFEGY MKALDIDFAT RKIAVRLTQT  41  L VIDQDGDNF K VKTTSTF FN  YDVDFTVGVE FDEYTKSLDN  81 RHVKALVTWE GDVLVCVQKG EKENRGWK KW IEGDKLYLEL 121 TCGDQVCRQV FKKK

A separate construct was made where the GFP-CRBP construct was fusedwith a nucleic acid encoding Retinoblastoma Protein (RB).

The GFP-CRBP and GFP-CRBP-RB constructs were separately transfected intocarcinoma cells, and the cells were observed using confocal microscopy.As shown in FIGS. 4B and 4C, the cells that fluoresce with green lightirradiation (from GFP emission) also fluoresce with red light,indicating that CRBP, which is excited (and emits) in the red region ofthe spectrum gives rise to significant fluorescence even when fused toGFP and even when present within cells. These data also furtherillustrate that the merocyanine dye can pass into these cells, bindspecifically to CRBP, and undergo fluorescence that is specificallycorrelated with the presence of the CRBP polypeptide.

Example 5 Fluorescent pH Sensor Useful in Living Cells

The Example describes the development of a protein-based fluorescent, pHsensor that can be used in living cells.

The CRABPII polypeptide sequence SEQ ID NO:30 was modified bymutagenesis of a nucleic acid including the SEQ ID NO:31 sequence toyield a modified nucleic acid encoding anR111K:R132Q:Y134F:T54V:R59W:A32W:M93L:E73A CRABPII polypeptide. Theseamino acid substitutions were selected by tuning the pKa of the Schiffbase because Schiff base protonation has a large effect on theabsorption of this system. The sequence of this modifiedR111K:R132Q:Y134F:T54V:R59W:A32W:M93L:E73A CRABPII polypeptide with theN-terminal methionine (SEQ ID NO:41) is shown below:

  1 MPNFSGNWKI IRSENFEELL KVLGVNVMLR KI W VAAASKP  41 AVEIKQEGDT FYIK VSTTV W  TTEINFKVGE EFE A QTVDGR  81 PCKSLVKWES ENK L VCEQKL LKGEGPKTSW TK ELTNDGEL 121 ILTMTADDVV CT Q V F VRE

The modified R111K:R132Q:Y134F:T54V:R59W:A32W:M93L:E73A CRABPIIpolypeptide without the N-terminal methionine is shown below (SEQ IDNO:42).

  1 PNFSGNWKII RSENFEELLK VLGVNVMLRK I W VAAASKPA  41 VEIKQEGDTF YIK VSTTV W T TEINFKVGEE FE A QTVDGRP  81 CKSLVKWESE NK L VCEQKLL KGEGPKTSWT K ELTNDGELI 121 LTMTADDVVC T Q V F VRE

As illustrated in FIG. 5, this modified CRABPII polypeptide acts as afluorescence-based pH sensor when combined with the merocyanine dyeshown in FIG. 5C. FIG. 5A demonstrates that the color of light absorbedby this protein changes dramatically with a change in pH. Thus, at pH11.25, the wavelength of maximum absorption is about 420 nm, but at pH7.3 the wavelength of maximum absorption is about 600 nm FIG. 5B showsthat the smallest absorption corresponds to the highest pH and thelowest absorption corresponds to the lowest pH. This ‘titration’ curvewas generated from the data shown in FIG. 5A. FIG. 5C shows fluorescenceemission spectra of the mutant CRABPII/merocyanine dye complex at pH 7.3(the highest emission) and pH 8.6 (the lowest emission). The structureof the merocyanine dye at the lower and higher pH is also shown.

FIG. 6 shows the light absorption and transmission properties of twoother mutant CRABPII/retinal complexes. The sequence of the firstmodified R111K:R132L:Y134F:T54V:R59W:A32W:M93:E73 CRABPII polypeptidewith the N-terminal methionine (SEQ ID NO:43) is shown below:

  1 MPNFSGNWKI IRSENFEELL KVLGVNVMLR KI W VAAASKP  41 AVEIKQEGDT FYIK VSTTV W  TTEINFKVGE EFE E QTVDGR  81 PCKSLVKWES ENK M VCEQKL LKGEGPKTSW TK ELTNDGEL 121 ILTMTADDVV CT L V F VRENote that this modified CRABPII polypeptide (SEQ ID NO:41) has wild typeamino acids at position 73 (E) and 93 (M).

The sequence of the second modifiedR111K:R132L:Y134F:T54V:R59Y:A32W:M93:E73 CRABPII polypeptide with theN-terminal methionine (SEQ ID NO:44) is shown below:

  1 MPNFSGNWKI IRSENFEELL KVLGVNVMLR KI W VAAASKP  41 AVEIKQEGDT FYIK VSTTV Y  TTEINFKVGE EFE E QTVDGR  81 PCKSLVKWES ENK M VCEQKL LKGEGPKTSW TK ELTNDGEL 121 ILTMTADDVV CT L V F VRE

Note that these modified CRABPII polypeptides have wild type amino acidsat position 73 (E) and 93 (M) and differ by only one amino acid atposition 59.

FIG. 6A shows that the first modified CRABPII polypeptide (SEQ ID NO:43)has a darker color (blue when seen in color) at pH 5.0 and a lightercolor (pale yellow when seen in color) at pH 7.3. FIG. 6B shows thatthis first modified CRAPII polypeptide has two strong absorption maximaat pH 5.0, one at about 375 nm and the other at about 610 nm. However,the absorption at about 610 nm of this first CRABPII polypeptide isgreatly reduced at pH 7.3.

FIG. 6C shows the absorption spectrum of a second modified CRAPIIpolypeptide (SEQ ID NO:44), which has two strong absorption maxima at pH5.0, one at about 375 nm and the other at about 590 nm. However, theabsorption at about 590 nm of this second modified CRABPII polypeptideis greatly reduced at pH 7.3. FIG. 6D shows that the second modifiedCRABPII polypeptide has a darker color (purple when seen in color) at pH5.0 and a lighter color (pale orange when seen in color) at pH 7.3.

Example 6 Colorimetric/Fluorescent Polypeptides are Stable Across WideChanges in Temperature and pH

This Example shows that modified CRABPII polypeptides are alsoremarkably stable across a wide range of pH and temperature conditions.

Thermostability studies on mutant CRABPII polypeptides were carried outusing circular dichroism (CD) measurements. Thermostability was assessedby observing a signal for properly folded proteins and loss of signalupon protein denaturation.

The behavior of two different CRABPII mutants was monitored as afunction of temperature change. In particular, a modified CRABPIIpolypeptide that exhibited good thermostability has amino acid sequenceSEQ ID NO:42. The thermostability of the SEQ ID NO:42 CRABPIIpolypeptide was compared to a R111K:R132L:Y134F CRABPII polypeptide withthe following sequence (SEQ ID NO:45).

  1 MPNFSGNWKI IRSENFEELL KVLGVNVMLR KIAVAAASKP 41 AVEIKQEGDT FYIKTSTTVR TTEINFKVGE EFEEQTVDGR 81 PCKSLVKWES ENKMVCEQKL LKGEGPKTSW T K ELTNDGEL 121 ILTMTADDVV CT L VF VRE

FIGS. 7 and 8 demonstrate that the mutant CRABPII polypeptide with SEQID NO:42 was remarkably stable across a wide range of pH and temperatureconditions. Thus, the structure of the CRABPII polypeptide can bemodified to optimize amino acid positioning and generate thermostableproteins that are also stable in acid and basic pH conditions.

These data illustrate that the mutant CRABPII/merocyanine dye complexcan emit fluorescence over a wide pH range, and therefore act aspH-sensor. Because the mutant CRABPII is a polypeptide that is readilyexpressed in living cells, and the merocyanine dye readily penetratesliving cells (see Examples 3 and 4), this system can be used as an invivo pH sensor. When fused to a selected biological product (e.g., afusion partner), the in vivo pH sensor can be used to sense pH changeswithin the biological product or in the microenvironment surrounding thebiological product.

All patents and publications referenced or mentioned herein areindicative of the levels of skill of those skilled in the art to whichthe invention pertains, and each such referenced patent or publicationis hereby specifically incorporated by reference to the same extent asif it had been incorporated by reference in its entirety individually orset forth herein in its entirety. Applicants reserve the right tophysically incorporate into this specification any and all materials andinformation from any such cited patents or publications.

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. The methods and processesillustratively described herein suitably may be practiced in differingorders of steps, and that they are not necessarily restricted to theorders of steps indicated herein or in the claims. As used herein and inthe appended claims, the singular forms “a,” “an,” and “the” includeplural reference unless the context clearly dictates otherwise. Thus,for example, a reference to “an antibody” includes a plurality (forexample, a solution of antibodies or a series of antibody preparations)of such antibodies, and so forth. Under no circumstances may the patentbe interpreted to be limited to the specific examples or embodiments ormethods specifically disclosed herein. Under no circumstances may thepatent be interpreted to be limited by any statement made by anyExaminer or any other official or employee of the Patent and TrademarkOffice unless such statement is specifically and without qualificationor reservation expressly adopted in a responsive writing by Applicants.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims and statements of theinvention.

The Abstract is provided to comply with 37 C.F.R. §1.72(b) to allow thereader to quickly ascertain the nature and gist of the technicaldisclosure. The Abstract is submitted with the understanding that itwill not be used to interpret or limit the scope or meaning of theclaims.

This application therefore discloses the following embodiments

1. An isolated nucleic acid encoding a modified polypeptide selectedfrom a member of the intracellular lipid binding protein family, whereinthe modified polypeptide transmits or emits light when bound to aretinoid or fluorescent dye molecule, and wherein the intracellularlipid binding protein has been modified so that an amino acid at any ofpositions 102-135 can form a Schiff base with a retinoid.2. The isolated nucleic acid of embodiment 1, which encodes a modifiedpolypeptide that has been modified by replacement of an amino acid atany of positions 102-135 with a lysine.3. The isolated nucleic acid of any of embodiments 1-21, which encodes amodified polypeptide that has been modified by replacement of aglutamine at any of amino acid positions 107, 108 or 109 with a lysine.4. The isolated nucleic acid of any of embodiments 1-21, which encodes amodified polypeptide that has been modified by replacement of anarginine at any of amino acid positions 110, 111 or 112 with a lysine.5. The isolated nucleic acid of any of embodiments 1-21, which encodes amodified polypeptide that has been modified by replacement of anarginine at any of amino acid positions 131, 132 or 133 with a lysine ora glutamine.6. The isolated nucleic acid of any of embodiments 1-21, which encodes amodified intracellular lipid binding protein that is further modified byreplacement of a lysine at any of amino acid positions 39, 40 or 41 witha leucine, serine or asparagine7. The isolated nucleic acid of any of embodiments 1-21, which encodes amodified intracellular lipid binding protein that is further modified byreplacement of a threonine at any of amino acid positions 50, 51, 52,53, 54 or 55 with an aspartic acid, asparagine, cysteine or a valine.8. The isolated nucleic acid of any of embodiments 1-21, which encodes amodified intracellular lipid binding protein that is further modified byreplacement of a tyrosine at any of amino acid positions 59, 60 or 61with a tryptophan, histidine, threonine, asparagine or phenylalanine.9. The isolated nucleic acid of any of embodiments 1-21, which encodes amodified intracellular lipid binding protein that is further modified byreplacement of an arginine at any of amino acid positions 57, 58, 59 or60 with a phenylalanine, tyrosine, tryptophan, leucine, glutamine,glutamic acid, aspartic acid or alanine.10. The isolated nucleic acid of any of embodiments 1-21, which encodesa modified intracellular lipid binding protein that is further modifiedby replacement of a tyrosine at any of amino acid positions 133, 134 or135 with a phenylalanine.11. The isolated nucleic acid of any of embodiments 1-21, which encodesa modified intracellular lipid binding protein that is further modifiedby replacement of a threonine at any of amino acid positions 28, 29 or30 with a leucine, tryptophan, glutamic acid or aspartic acid.12. The isolated nucleic acid of any of embodiments 1-21, which encodesa modified intracellular lipid binding protein that is further modifiedby replacement of an alanine at any of amino acid positions 30, 31, 32or 33 with a tryptophan, phenylalanine, tyrosine, serine, histidine,glutamic acid or leucine.13. The isolated nucleic acid of any of embodiments 1-21, which encodesa modified intracellular lipid binding protein that is further modifiedby replacement of a tyrosine at any of amino acid positions 18, 19 or 20with a tryptophan or phenyalanine.14. The isolated nucleic acid of any of embodiments 1-21, which encodesa modified intracellular lipid binding protein that is further modifiedby replacement of a glutamine at any of amino acid positions 3, 4 or 5with an arginine, asparagine, phenylalanine, leucine, alanine,tryptophan, threonine, glutamic acid, histidine, or lysine.15. The isolated nucleic acid of any of embodiments 1-21, which encodesa modified intracellular lipid binding protein that is further modifiedby replacement of a methionine at any of amino acid positions 92, 93 or94 with a leucine.16. The isolated nucleic acid of any of embodiments 1-21, which encodesa modified intracellular lipid binding protein that is further modifiedby replacement of a glutamic acid at any of amino acid positions 72, 73or 74 with an alanine or leucine.17. The isolated nucleic acid of any of embodiments 1-21, which encodesa modified intracellular lipid binding protein that is further modifiedby replacement of a glutamine at any of amino acid positions 36, 37 or38 with a leucine, methionine or tryptophan.18. The isolated nucleic acid of any of embodiments 1-21, which encodesa modified intracellular lipid binding protein that is further modifiedby replacement of a glutamine at any of amino acid positions 128, 129 or130 with a leucine, lysine, glutamic acid or tryptophan.19. The isolated nucleic acid of any of embodiments 1-21, wherein themodified intracellular lipid binding protein is a modified cellularretinoic acid binding protein II (CRABPII) or a modified cellularretinol binding protein II (CRBPII).20. The isolated nucleic acid of any of embodiments 1-19, encoding amodified CRABPII polypeptide with amino acid sequence SEQ ID NO:46:

  1 MPNXSGNWKX IRXENXEELX KVLGXNVMLR KIXVAXXXXX 41 AVEIKXEGDT FYIKXSXXXX TXEINFKVGE EFEXXTXDXR 81 PXKSLVKWES ENKXVXEQKL LKGEGPKTSW TKELTNDGEL 121 IXTXTADDVV XTXVXVREwherein each X is independently a genetically encoded L-amino acid, anaturally-occurring non-genetically encoded L-amino acid, a syntheticL-amino acid or a synthetic D-amino acid.21. The isolated nucleic acid of any of embodiments 1-19, encoding amodified CRBPII polypeptide with amino acid SEQ ID NO:47:

  1 TXDXNGTWEM ESNENXEGXX KALDXDFAXR KIXVRLTXTX 41 VXDQDGDNFK XKXTXTXXNX DXDXTVGVEF DXYTKXXDNR 81 HVKALVTWEG DVLVXVXKGE KENXGXKXWI EGDKLYXEXT 121 CGDQVCRXVX KKKwherein each X is independently a genetically encoded L-amino acid, anaturally-occurring non-genetically encoded L-amino acid, a syntheticL-amino acid or a synthetic D-amino acid.22. A hybrid nucleic acid comprising the isolated nucleic acid of any ofembodiments 1-21 joined to a fusion partner nucleic acid that encodes afusion partner polypeptide.23. The hybrid nucleic acid of embodiment 22, wherein the isolatednucleic acid is joined in frame to the fusion partner nucleic acid.24. An expression cassette comprising the isolated nucleic acid of anyof embodiments 1-21 and at least one nucleic acid segment encoding aregulatory element.25. A vector comprising the isolated nucleic acid of any of embodiments1-21.26. A vector comprising the expression cassette of embodiment 25.27. A host cell comprising the isolated nucleic acid of any ofembodiments 1-28.28. The host cell of embodiment 27, wherein the isolated nucleic acid iswithin an expression cassette, a vector or a combination thereof.29. A modified polypeptide selected from a member of the intracellularlipid binding protein family, wherein the modified polypeptide transmitsor emits light when bound to a retinoid or fluorescent dye molecule, andwherein the intracellular lipid binding protein has been modified sothat an amino acid at any of positions 102-135 can form a Schiff basewith a retinoid.30. The modified polypeptide of embodiment 29, which has been modifiedby replacement of the amino acid at any of positions 102-135 with alysine.31. The modified polypeptide of any of embodiments 29-50, which at hasbeen modified by replacement of a glutamine at any of amino acidpositions 107, 108 or 109 with a lysine.32. The modified polypeptide of any of embodiments 29-50, which has beenmodified by replacement of an arginine at any of amino acid positions110, 111 or 112 with a lysine.33. The modified polypeptide of any of embodiments 29-50, which has beenmodified by replacement of an arginine at any of amino acid positions131, 132 or 133 with a lysine or a glutamine.34. The modified polypeptide of any of embodiments 29-50, which isfurther modified by replacement of a lysine at any of amino acidpositions 39, 40 or 41 with a leucine, serine or asparagine.35. The modified polypeptide of any of embodiments 29-50, which isfurther modified by replacement of a threonine at any of amino acidpositions 50, 51, 52, 53, 54 or 55 with an aspartic acid, asparagine,cysteine or a valine.36. The modified polypeptide of any of embodiments 29-50, which isfurther modified by replacement of a tyrosine at any of amino acidpositions 59, 60 or 61 with a tryptophan, histidine, threonine,asparagine or phenylalanine37. The modified polypeptide of any of embodiments 29-50, which isfurther modified by replacement of an arginine at any of amino acidpositions 57, 58, 59 or 60 with a phenylalanine, tyrosine, tryptophan,leucine, glutamine, glutamic acid, aspartic acid or alanine.38. The modified polypeptide of any of embodiments 29-50, which isfurther modified by replacement of a tyrosine at any of amino acidpositions 133, 134 or 135 with a phenylalanine.39. The modified polypeptide of any of embodiments 29-50, which isfurther modified by replacement of a threonine at any of amino acidpositions 28, 29 or 30 with a leucine, tryptophan, glutamic acid oraspartic acid.40. The modified polypeptide of any of embodiments 29-50, which isfurther modified by replacement of an alanine at any of amino acidpositions 30, 31, 32 or 33 with a tryptophan, phenylalanine, tyrosine,serine, histidine, glutamic acid or leucine.41. The modified polypeptide of any of embodiments 29-50, which isfurther modified by replacement of a tyrosine at any of amino acidpositions 18, 19 or 20 with a tryptophan or phenylalanine.42. The modified polypeptide of any of embodiments 29-50, which isfurther modified by replacement of a glutamine at any of amino acidpositions 3, 4 or 5 with an arginine, asparagine, phenylalanine,leucine, alanine, tryptophan, threonine, glutamic acid, histidine, orlysine.43. The modified polypeptide of any of embodiments 29-50, which isfurther modified by replacement of a methionine at any of amino acidpositions 92, 93 or 94 with a leucine.44. The modified polypeptide of any of embodiments 29-50, which isfurther modified by replacement of a glutamic acid at any of amino acidpositions 72, 73 or 74 with an alanine or leucine.45. The modified polypeptide of any of embodiments 29-50, which isfurther modified by replacement of a glutamine at any of amino acidpositions 36, 37 or 38 with a leucine, methionine or tryptophan.46. The modified polypeptide of any of embodiments 29-50, which isfurther modified by replacement of a glutamine at any of amino acidpositions 128, 129 or 130 with a leucine, lysine, glutamic acid ortryptophan.47. The modified polypeptide of any of embodiments 29-50, which is amodified cellular retinoic acid binding protein II (CRABPII) or amodified cellular retinol binding protein II (CRBPII).48. The modified polypeptide of embodiment 29, which comprises an aminoacid sequence selected from the group consisting of SEQ ID NO:6-28,39-47, or a combination thereof.49. The modified polypeptide of any of embodiments 29-50, whichcomprises a modified CRABPII amino acid sequence SEQ ID NO:46:

  1 MPNXSGNWKX IRXENXEELX KVLGXNVMLR KIXVAXXXXX 41 AVEIKXEGDT FYIKXSXXXX TXEINFKVGE EFEXXTXDXR 81 PXKSLVKWES ENKXVXEQKL LKGEGPKTSW TKELTNDGEL 121 IXTXTADDVV XTXVXVREwherein each X is independently a genetically encoded L-amino acid, anaturally-occurring non-genetically encoded L-amino acid, a syntheticL-amino acid or a synthetic D-amino acid.50. The modified polypeptide of any of embodiments 29-50, whichcomprises a modified CRBPII amino acid sequence SEQ ID NO:47:

  1 TXDXNGTWEM ESNENXEGXX KALDXDFAXR KIXVRLTXTX 41 VXDQDGDNFK XKXTXTXXNX DXDXTVGVEF DXYTKXXDNR 81 HVKALVTWEG DVLVXVXKGE KENXGXKXWI EGDKLYXEXT 121 CGDQVCRXVX KKKwherein each X is independently a genetically encoded L-amino acid, anaturally-occurring non-genetically encoded L-amino acid, a syntheticL-amino acid or a synthetic D-amino acid.51. The modified polypeptide of any of embodiments 29-50, which is mixedwith or complexed with a retinoid or dye ligand.52. A fusion protein comprising the modified polypeptide of any ofembodiments 29-51 fused to another protein.53. A kit comprising at least one container comprising the isolatednucleic acid of any of embodiments 1-21, and a second containercomprising a retinoid or dye ligand that binds a modified polypeptideencoded by the isolated nucleic acid, wherein the isolated nucleic acidcan be within an expression cassette or vector.54. A kit comprising at least one container comprising the modifiedpolypeptide of any of embodiments 29-50 and a second containercomprising a retinoid or dye ligand that binds a modified polypeptide.55. A method of observing a target protein in vivo comprising contactinga living cell with a retinoid or dye ligand that binds a modifiedpolypeptide encoded by the isolated nucleic acid of any of embodiments1-21, wherein the cell expresses a fusion protein comprising themodified polypeptide fused in frame with the target protein.56. The method of embodiment 55, wherein the dye ligand is a compound offormula I:

Ring-Y—CHO

wherein:

-   -   Ring is an optionally substituted C₅-C₁₄ mono-, di- or tricyclic        cycloalkyl, aryl or heterocyclic ring, wherein the heterocyclic        ring has at least one nitrogen or oxygen ring atom, and wherein        the Ring has 1-3 optional substituents that are selected from        the group consisting of alkyl, halogen, alkoxy, amino and        sulfhydryl; and

Y is a divalent C₂-C₁₂ alkenylene chain that optionally substituted with1-3 alkyl groups.

57. The method of embodiment 55, wherein the retinoid is retinal.58. A method of making a colorimetric and/or fluorescent proteincomprising modifying a nucleic acid encoding an intracellular lipidbinding protein family member to generate a modified iLBP polypeptidewherein the modified iLBP polypeptide transmits or emits light whenbound to a retinoid or fluorescent dye molecule, and wherein theintracellular lipid binding protein has been modified so that an aminoacid at any of positions 102-135 can form a Schiff base with a retinoid(e.g., retinal).59. The method of embodiment 58, wherein the nucleic is modified toencode the modified polypeptide of any of embodiments 29-50.60. The method of embodiment 58 or 59 further comprising contacting themodified polypeptide with a retinal or dye ligand.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

1. An isolated nucleic acid encoding a modified polypeptide selectedfrom a member of the intracellular lipid binding protein family, whereinthe modified polypeptide transmits or emits light when bound to aretinoid or fluorescent dye molecule, and wherein the intracellularlipid binding protein has been modified so that an amino acid at any ofpositions 102-135 can form a Schiff base with a retinoid.
 2. Theisolated nucleic acid of claim 1, which encodes a modified polypeptidethat has been modified by replacement of an amino acid at any ofpositions 102-135 with a lysine.
 3. The isolated nucleic acid of claim1, which encodes a modified polypeptide that has been modified byreplacement of a glutamine at any of amino acid positions 107, 108 or109 with a lysine.
 4. The isolated nucleic acid of claim 1, whichencodes a modified polypeptide that has been modified by replacement ofan arginine at any of amino acid positions 110, 111 or 112 with alysine.
 5. The isolated nucleic acid of claim 1, which encodes amodified polypeptide that has been modified by replacement of anarginine at any of amino acid positions 131, 132 or 133 with a lysine ora glutamine.
 6. The isolated nucleic acid of claim 1, which encodes amodified intracellular lipid binding protein that is further modified byreplacement of a lysine at any of amino acid positions 39, 40 or 41 witha leucine, serine or asparagine.
 7. The isolated nucleic acid of claim1, which encodes a modified intracellular lipid binding protein that isfurther modified by replacement of a threonine at any of amino acidpositions 50, 51, 52, 53, 54 or 55 with an aspartic acid, asparagine,cysteine or a valine.
 8. The isolated nucleic acid of claim 1, whichencodes a modified intracellular lipid binding protein that is furthermodified by replacement of a tyrosine at any of amino acid positions 59,60 or 61 with a tryptophan, histidine, threonine, asparagine orphenylalanine.
 9. The isolated nucleic acid of claim 1, which encodes amodified intracellular lipid binding protein that is further modified byreplacement of an arginine at any of amino acid positions 57, 58, 59 or60 with a phenylalanine, tyrosine, tryptophan, leucine, glutamine,glutamic acid, aspartic acid or alanine.
 10. The isolated nucleic acidof claim 1, which encodes a modified intracellular lipid binding proteinthat is further modified by replacement of a tyrosine at any of aminoacid positions 133, 134 or 135 with a phenylalanine.
 11. The isolatednucleic acid of claim 1, which encodes a modified intracellular lipidbinding protein that is further modified by replacement of a threonineat any of amino acid positions 28, 29 or 30 with a leucine, tryptophan,glutamic acid or aspartic acid.
 12. The isolated nucleic acid of claim1, which encodes a modified intracellular lipid binding protein that isfurther modified by replacement of an alanine at any of amino acidpositions 30, 31, 32 or 33 with a tryptophan, phenylalanine, tyrosine,serine, histidine, glutamic acid or leucine.
 13. The isolated nucleicacid of claim 1, which encodes a modified intracellular lipid bindingprotein that is further modified by replacement of a tyrosine at any ofamino acid positions 18, 19 or 20 with a tryptophan or phenyalanine. 14.The isolated nucleic acid of claim 1, which encodes a modifiedintracellular lipid binding protein that is further modified byreplacement of a glutamine at any of amino acid positions 3, 4 or 5 withan arginine, asparagine, phenylalanine, leucine, alanine, tryptophan,threonine, glutamic acid, histidine, or lysine.
 15. The isolated nucleicacid of claim 1, which encodes a modified intracellular lipid bindingprotein that is further modified by replacement of a methionine at anyof amino acid positions 92, 93 or 94 with a leucine.
 16. The isolatednucleic acid of claim 1, which encodes a modified intracellular lipidbinding protein that is further modified by replacement of a glutamicacid at any of amino acid positions 72, 73 or 74 with an alanine orleucine.
 17. The isolated nucleic acid of claim 1, which encodes amodified intracellular lipid binding protein that is further modified byreplacement of a glutamine at any of amino acid positions 36, 37 or 38with a leucine, methionine or tryptophan.
 18. The isolated nucleic acidof claim 1, which encodes a modified intracellular lipid binding proteinthat is further modified by replacement of a glutamine at any of aminoacid positions 128, 129 or 130 with a leucine, lysine, glutamic acid ortryptophan.
 19. The isolated nucleic acid of claim 1, wherein themodified intracellular lipid binding protein is a modified cellularretinoic acid binding protein II (CRABPII) or a modified cellularretinol binding protein II (CRBPII).
 20. The isolated nucleic acid ofclaim 1, encoding a modified CRABPII polypeptide with amino acidsequence SEQ ID NO:46:   1 MPNXSGNWKX IRXENXEELX KVLGXNVMLR KIXVAXXXXX 41 AVEIKXEGDT FYIKXSXXXX TXEINFKVGE EFEXXTXDXR 81 PXKSLVKWES ENKXVXEQKL LKGEGPKTSW TKELTNDGEL 121 IXTXTADDVV XTXVXVRE

wherein each X is independently a genetically encoded L-amino acid, anaturally-occurring non-genetically encoded L-amino acid, a syntheticL-amino acid or a synthetic D-amino acid.
 21. The isolated nucleic acidof claim 1, encoding a modified CRBPII polypeptide with amino acid SEQID NO:47:   1 TXDXNGTWEM ESNENXEGXX KALDXDFAXR KIXVRLTXTX 41 VXDQDGDNFK XKXTXTXXNX DXDXTVGVEF DXYTKXXDNR 81 HVKALVTWEG DVLVXVXKGE KENXGXKXWI EGDKLYXEXT 121 CGDQVCRXVX KKK

wherein each X is independently a genetically encoded L-amino acid, anaturally-occurring non-genetically encoded L-amino acid, a syntheticL-amino acid or a synthetic D-amino acid.
 22. A hybrid nucleic acidcomprising the isolated nucleic acid of claim 1 joined to a fusionpartner nucleic acid that encodes a fusion partner polypeptide.
 23. Thehybrid nucleic acid of claim 22, wherein the isolated nucleic acid isjoined in frame to the fusion partner nucleic acid.
 24. An expressioncassette comprising the isolated nucleic acid of claim 1 and at leastone nucleic acid segment encoding a regulatory element.
 25. A vectorcomprising the isolated nucleic acid of claim
 1. 26. A vector comprisingthe expression cassette of claim
 25. 27. A host cell comprising theisolated nucleic acid of claim
 1. 28. The host cell of claim 27, whereinthe isolated nucleic acid is within an expression cassette, a vector ora combination thereof.
 29. A modified polypeptide selected from a memberof the intracellular lipid binding protein family, wherein the modifiedpolypeptide transmits or emits light when bound to a retinoid orfluorescent dye molecule, and wherein the intracellular lipid bindingprotein has been modified so that an amino acid at any of positions102-135 can form a Schiff base with a retinoid.
 30. The modifiedpolypeptide of claim 29, which has been modified by replacement of theamino acid at any of positions 102-135 with a lysine.
 31. The modifiedpolypeptide of claim 29, which at has been modified by replacement of aglutamine at any of amino acid positions 107, 108 or 109 with a lysine.32. The modified polypeptide of claim 29, which has been modified byreplacement of an arginine at any of amino acid positions 110, 111 or112 with a lysine.
 33. The modified polypeptide of claim 29, which hasbeen modified by replacement of an arginine at any of amino acidpositions 131, 132 or 133 with a lysine or a glutamine.
 34. The modifiedpolypeptide of claim 29, which is further modified by replacement of alysine at any of amino acid positions 39, 40 or 41 with a leucine,serine or asparagine.
 35. The modified polypeptide of claim 29, which isfurther modified by replacement of a threonine at any of amino acidpositions 50, 51, 52, 53, 54 or 55 with an aspartic acid, asparagine,cysteine or a valine.
 36. The modified polypeptide of claim 29, which isfurther modified by replacement of a tyrosine at any of amino acidpositions 59, 60 or 61 with a tryptophan, histidine, threonine,asparagine or phenylalanine
 37. The modified polypeptide of claim 29,which is further modified by replacement of an arginine at any of aminoacid positions 57, 58, 59 or 60 with a phenylalanine, tyrosine,tryptophan, leucine, glutamine, glutamic acid, aspartic acid or alanine.38. The modified polypeptide of claim 29, which is further modified byreplacement of a tyrosine at any of amino acid positions 133, 134 or 135with a phenylalanine.
 39. The modified polypeptide of claim 29, which isfurther modified by replacement of a threonine at any of amino acidpositions 28, 29 or 30 with a leucine, tryptophan, glutamic acid oraspartic acid.
 40. The modified polypeptide of claim 29, which isfurther modified by replacement of an alanine at any of amino acidpositions 30, 31, 32 or 33 with a tryptophan, phenylalanine, tyrosine,serine, histidine, glutamic acid or leucine.
 41. The modifiedpolypeptide of claim 29, which is further modified by replacement of atyrosine at any of amino acid positions 18, 19 or 20 with a tryptophanor phenylalanine.
 42. The modified polypeptide of claim 29, which isfurther modified by replacement of a glutamine at any of amino acidpositions 3, 4 or 5 with an arginine, asparagine, phenylalanine,leucine, alanine, tryptophan, threonine, glutamic acid, histidine, orlysine.
 43. The modified polypeptide of claim 29, which is furthermodified by replacement of a methionine at any of amino acid positions92, 93 or 94 with a leucine.
 44. The modified polypeptide of claim 29,which is further modified by replacement of a glutamic acid at any ofamino acid positions 72, 73 or 74 with an alanine or leucine.
 45. Themodified polypeptide of claim 29, which is further modified byreplacement of a glutamine at any of amino acid positions 36, 37 or 38with a leucine, methionine or tryptophan.
 46. The modified polypeptideof claim 29, which is further modified by replacement of a glutamine atany of amino acid positions 128, 129 or 130 with a leucine, lysine,glutamic acid or tryptophan.
 47. The modified polypeptide of claim 29,which is a modified cellular retinoic acid binding protein II (CRABPII)or a modified cellular retinol binding protein II (CRBPII).
 48. Themodified polypeptide of claim 29, which comprises an amino acid sequenceselected from the group consisting of SEQ ID NO:6-28, 39-47, or acombination thereof.
 49. The modified polypeptide of claim 29, whichcomprises a modified CRABPII amino acid sequence SEQ ID NO:46:  1 MPNXSGNWKX IRXENXEELX KVLGXNVMLR KIXVAXXXXX 41 AVEIKXEGDT FYIKXSXXXX TXEINFKVGE EFEXXTXDXR 81 PXKSLVKWES ENKXVXEQKL LKGEGPKTSW TKELTNDGEL 121 IXTXTADDVV XTXVXVRE

wherein each X is independently a genetically encoded L-amino acid, anaturally-occurring non-genetically encoded L-amino acid, a syntheticL-amino acid or a synthetic D-amino acid.
 50. The modified polypeptideof claim 29, which comprises a modified CRBPII amino acid sequence SEQID NO:47:   1 TXDXNGTWEM ESNENXEGXX KALDXDFAXR KIXVRLTXTX 41 VXDQDGDNFK XKXTXTXXNX DXDXTVGVEF DXYTKXXDNR 81 HVKALVTWEG DVLVXVXKGE KENXGXKXWI EGDKLYXEXT 121 CGDQVCRXVX KKK

wherein each X is independently a genetically encoded L-amino acid, anaturally-occurring non-genetically encoded L-amino acid, a syntheticL-amino acid or a synthetic D-amino acid.
 51. The modified polypeptideof claim 29, which is mixed with or complexed with a retinoid or dyeligand.
 52. A fusion protein comprising the modified polypeptide ofclaim 29 fused to another protein.
 53. A kit comprising at least onecontainer comprising the isolated nucleic acid of claim 1, and a secondcontainer comprising a retinoid or dye ligand that binds a modifiedpolypeptide encoded by the isolated nucleic acid, wherein the isolatednucleic acid can be within an expression cassette or vector.
 54. A kitcomprising at least one container comprising the modified polypeptide ofclaim 29 and a second container comprising a retinoid or dye ligand thatbinds a modified polypeptide.
 55. A method of observing a target proteinin vivo comprising contacting a living cell with a retinoid or dyeligand that binds a modified polypeptide encoded by the isolated nucleicacid of claim 1, wherein the cell expresses a fusion protein comprisingthe modified polypeptide fused in frame with the target protein.
 56. Themethod of claim 55, wherein the dye ligand is a compound of formula I:Ring-Y—CHO wherein: Ring is an optionally substituted C₅-C₁₄ mono-, di-or tricyclic cycloalkyl, aryl or heterocyclic ring, wherein theheterocyclic ring has at least one nitrogen or oxygen ring atom, andwherein the Ring has 1-3 optional substituents that are selected fromthe group consisting of alkyl, halogen, alkoxy, amino and sulfhydryl;and Y is a divalent C₂-C₁₂ alkenylene chain that optionally substitutedwith 1-3 alkyl groups.
 57. The method of claim 55, wherein the retinoidis retinal.